Methods and pharmaceutical compositions for the treatment and prevention of hepatitis C infection

ABSTRACT

This invention relates to cyclosporin derivatives of general formula (I):  
                 
 
wherein A, B, R 1 , R 2  and X are as defined in the specification, and pharmaceutical compositions prepared from the same, for use in treatment of hepatitis C virus.

This application claims priority to U.S. provisional application Ser.Nos. 60/722,679, filed Sep. 30, 2005, entitled “METHODS ANDPHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT AND PREVENTION OFHEPATITIS C INFECTION” to David Houck and 60/787,549, filed Mar. 29,2006, entitled “METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THETREATMENT AND PREVENTION OF HEPATITIS C INFECTION” to David Houck. Thedisclosure of the above referenced applications is incorporated byreference herein.

1. FIELD OF THE INVENTION

The present invention provides methods and pharmaceutical compositions,for use in treatment or prevention of hepatitis C virus infection in asubject in need thereof. In certain aspects, the present inventionprovides methods of treating hepatitis C infection by administering to asubject in need thereof an amount of a 3-ether or 3-thioethercyclosporin of the invention in combination with a second agent inamounts effective to treat or prevent the infection.

2. BACKGROUND

In 1989, a main causative virus of non-A non-B post-transfusionhepatitis was found and named hepatitis C virus (HCV). Since then,several types of hepatitis viruses have been found besides type A, typeB and type C, wherein hepatitis caused by HCV is called hepatitis C.Subjects infected with HCV are considered to involve several percent ofthe world population, and infection with HCV characteristically becomeschronic.

HCV is an envelope RNA virus, wherein the genome is a single strandplus-strand RNA, and belongs to the genus Hepacivirus of Flavivirus(from The International Committee on Taxonomy of Viruses, InternationalUnion of Microbiological Societies). Of the same hepatitis viruses, forexample, hepatitis B virus (HBV), which is a DNA virus, is eliminated bythe immune system, and infection with this virus ends in an acuteinfection except for neonates and infants having yet immatureimmunological competence. In contrast, HCV somehow avoids the immunesystem of the host due to an unknown mechanism. Once infected with thisvirus, even an adult having a mature immune system frequently developspersistent infection.

When chronic hepatitis is associated with the persistent infection withHCV, it advances to cirrhosis or hepatic cancer in a high rate.Enucleation of tumor by operation does not help much, because thesubject often develops recurrent hepatic cancer due to the sequelainflammation in non-cancerous parts.

Thus, an effective therapeutic method for treating hepatitis C infectionis desired. Apart from the symptomatic therapy to suppress inflammationwith an anti-inflammatory agent, the development of a therapeutic agentthat reduces HCV to a low level free from inflammation and thateradicates HCV has been strongly demanded. An optimal therapeutic agentwould provide a virologic response classified as a “sustained virologicresponse,” which is defined as undetectable levels of virus in blood sixmonths or more after completing hepatitis C therapy.

At present, treatments with interferon, as a single agent or incombination with ribavirin, are the only effective methods known for theeradication of HCV. However, interferon can eradicate the virus only inabout 33-46% of the subject population. For the rest of the subjects, ithas no effect or provides only a temporary effect. Therefore, ananti-HCV drug to be used in the place of or concurrently with interferonis awaited in great expectation.

Cyclosporin A is well known for its immunosuppressive activity and arange of therapeutic uses, including antifungal, anti-parasitic, andanti-inflammatory as well as anti-HIV activity. Cyclosporin A andcertain derivatives have been reported as having anti-HCV activity, seeWatashi et al., 2003, Hepatology 38:1282-1288, Nakagawa et al., 2004,Biochem. Biophys. Res. Commun. 313:42-7, and Shimotohno and Watashi,2004, American Transplant Congress, Abstract No. 648 (American Journalof Transplantation, 2004, 4(s8):1-653).

However, a problem with known cyclosporins is their nephrotoxicity. Forexample, cyclosporin A (cyclosporine) can cause nephrotoxicity andhepatotoxicity. Nephrotoxicity, a serious complication of cyclosporinetherapy, is characterized by intense renal vasoconstriction that oftenprogresses to chronic injury with irreversible structural renal damage(Busauschina et al., 2004 Transplant Proc. 36: pages 229S-233S, andMyers BD and Newton L., J Am Soc Nephrol. 1991, (2 Supp. 1), pagesS45-52). Nephrotoxicity associated with cyclosporine has been noted in25 to 38% of transplant subjects. Renal dysfunction can occur at anytime and ranges from an early reversible damage to a late progression toirreversible chronic renal failure. Acute nephrotoxicity may appear soonafter transplantation or after weeks or months, with oliguria, acutedecrement of glomerular filtration rate and renal plasma flow (Kahan,1989).

In prolonged cyclosporine administration, chronic nephrotoxicity ischaracterized by a progressive and mostly irreversible impairment ofrenal function, and it is supported by histological lesions ranging fromstriped fibrosis to ischemic collapse of the tuft, glomerular sclerosisand tubular atrophy.

Effective methods and compositions for the treatment or prevention ofhepatitis C infection are needed to combat the virus worldwide.

3. SUMMARY OF THE INVENTION

It has found that certain 3-substituted cyclosporin derivatives haveactivity against HCV. Also, it has been found that certain of said3-substituted cyclosporin derivatives have unexpectedly goodtoxicological profiles, either alone or in combination with a secondagent. Accordingly, the present invention provides methods of treatingor preventing HCV infection with the 3-substituted cyclosporinderivatives of the invention along with a second agent effective fortreating or preventing HCV infection. The present invention alsoprovides pharmaceutical compositions for use in the methods.

In one aspect, the present invention provides the use of a 3-ether or3-thioether cyclosporin derivative of the invention along with a secondtherapeutic useful for the treatment or prevention of HCV infection.Exemplary therapeutic agents are described in detail in the sectionsbelow.

In another aspect, the present invention provides pharmaceuticalcompositions, single unit dosage forms, and kits suitable for use intreating or preventing HCV infection which comprise a therapeutically orprophylactically effective amount of 3-ether or 3-thioether cyclosporinderivative and a therapeutically or prophylactically effective amount ofa second therapeutic useful for the treatment or prevention of HCVinfection.

In certain embodiments, the 3-substituted cyclosporin derivative of theinvention is selected from the group consisting of a 3-ethercyclosporin; a 3-ether, 4-gamma-hydroxymethylleucine cyclosporin; a3-thioether cyclosporin; and a 3-thioether, 4-gamma-hydroxymethylleucinecyclosporin. In particular embodiments, the 3-substituted cyclosporinderivative is according to general formula (I):

wherein:A is residue of formula (IIa) or (IIb):

B is ethyl, 1-hydroxyethyl, isopropyl or n-propyl;R¹ represents:

-   -   straight- or branched-chain alkyl containing from one to six        carbon atoms, optionally substituted by one or more groups R³        which may be the same or different;    -   straight- or branched-chain alkenyl containing from two to six        carbon atoms optionally substituted by one or more groups which        may be the same or different selected from the group consisting        of halogen, hydroxy, amino, monoalkylamino and dialkylamino;    -   straight- or branched-chain alkynyl containing from two to six        carbon atoms, optionally substituted by one or one or more        groups which may be the same or different selected from the        group consisting of halogen, hydroxy, amino, monoalkylamino and        dialkylamino;    -   cycloalkyl containing from three to six carbon atoms optionally        substituted by one or more groups which may be the same or        different selected from the group consisting of halogen,        hydroxy, amino, monoalkylamino and dialkylamino;    -   straight- or branched-chain alkoxycarbonyl containing from one        to six carbon atoms;        R² represents isobutyl or 2-hydroxyisobutyl;        X represents —S(O)_(n)— or oxygen;        R³ is selected from the group consisting of halogen, hydroxy,        carboxyl, alkoxy, alkoxycarbonyl, —NR⁴R⁵ and —NR⁶(CH₂)_(m)NR⁴R⁵;        R⁴ and R⁵, which may be the same or different, represent:    -   hydrogen;    -   straight- or branched-chain alkyl comprising from one to six        carbon atoms, optionally substituted by one or more groups R⁷        which may be the same or different;    -   straight- or branched-chain alkenyl or alkynyl comprising from        two to four carbon atoms;    -   cycloalkyl containing from three to six carbon atoms optionally        substituted by straight- or branched-chain alkyl containing from        one to six carbon atoms;    -   phenyl optionally substituted by from one to five groups which        may be the same or different selected from the group consisting        of halogen, alkoxy, alkoxycarbonyl, amino, alkylamino and        dialkylamino;    -   a heterocyclic ring which may be saturated or unsaturated        containing five or six ring atoms and from one to three        heteroatoms which may the same or different selected from        nitrogen, sulfur and oxygen;    -   or R⁴ and R⁵, together with the nitrogen atom to which they are        attached, form a saturated or unsaturated heterocyclic ring        containing from four to six ring atoms, which ring may        optionally contain another heteroatom selected from the group        consisting of nitrogen, oxygen and sulfur and may be optionally        substituted by from one to four groups which may be the same or        different selected from the group consisting of alkyl, phenyl        and benzyl;        R⁶ represents hydrogen or straight- or branched-chain alkyl        containing from one to six carbon atoms;        R⁷ is selected from the group consisting of halogen, hydroxy,        carboxyl, alkoxycarbonyl and —NR⁸R⁹;        R⁸ and R⁹ which may be the same or different, each represent        hydrogen or straight- or branched-chain alkyl containing from        one to six carbon atoms;        n is zero, one or two;        m is an integer from two to four;        halogen means fluoro, chloro, bromo or iodo;        or a pharmaceutically acceptable salt thereof.

In certain cases the substituents A, B, R¹ and R² may contribute tooptical and/or stereoisomerism. All such forms are embraced by thepresent invention.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A provides the antiviral synergy volume for a combination ofCompound O and IFNα.

FIG. 1B provides the antiviral synergy volume for a combination ofCompound T and IFNα.

FIG. 1C provides the antiviral synergy volume for a combination ofCyclosporin A and IFNα.

FIG. 2 provides the antiviral synergy volume for a combination ofCompound O and 2′-C-methyl-cytidine.

FIG. 3A provides the antiviral synergy volume for a combination ofCompound O and Compound 3.

FIG. 3B provides the cytotoxicity volume for a combination of Compound Oand Compound 3.

5. DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present invention provides methods of treating or preventinghepatitis C infection in a subject in need thereof, and pharmaceuticalcompositions and dosage forms useful for such methods. The methods andcompositions are described in detail in the sections below.

5.1 Definitions

When referring to the compounds and complexes of the invention, thefollowing terms have the following meanings unless indicated otherwise.

“Cyclosporin”, refers to any cyclosporin compound known to those ofskill in the art, or a derivative thereof. See, e.g., Ruegger et al.,1976, Helv. Chim. Acta. 59:1075-92; Borel et al., 1977, Immunology32:1017-25; the contents of which are hereby incorporated by referencein their entireties. Exemplary compounds of the invention arecyclosporin derivatives. Unless noted otherwise, a cyclosporin describedherein is a cyclosporin A, and a cyclosporin derivative described hereinis a derivative of cyclosporin A.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 11 carbon atoms, more particularly as alower alkyl, from 1 to 8 carbon atoms and still more particularly, from1 to 6 carbon atoms. The hydrocarbon chain may be eitherstraight-chained or branched. This term is exemplified by groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl,n-hexyl, n-octyl, tert-octyl and the like. The term “lower alkyl” refersto alkyl groups having 1 to 6 carbon atoms.

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 11 carbon atoms and more particularly 1to 6 carbon atoms which can be straight-chained or branched. This termis exemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Alkenyl” refers to monovalent olefinically unsaturated hydrocarbylgroups preferably having up to about 11 carbon atoms, particularly, from2 to 8 carbon atoms, and more particularly, from 2 to 6 carbon atoms,which can be straight-chained or branched and having at least 1 andparticularly from 1 to 2 sites of olefinic unsaturation. Particularalkenyl groups include ethenyl (—CH═CH₂), n-propenyl (—CH₂CH═CH₂),isopropenyl (—C(CH₃)═CH₂), vinyl and substituted vinyl, and the like.

“Alkenylene” refers to divalent olefinically unsaturated hydrocarbylgroups particularly having up to about 11 carbon atoms and moreparticularly 2 to 6 carbon atoms which can be straight-chained orbranched and having at least 1 and particularly from 1 to 2 sites ofolefinic unsaturation. This term is exemplified by groups such asethenylene (—CH═CH—), the propenylene isomers (e.g., —CH═CHCH₂— and—C(CH₃)═CH— and —CH═C(CH₃)—) and the like.

“Alkynyl” refers to acetylenically unsaturated hydrocarbyl groupsparticularly having up to about 11 carbon atoms and more particularly 2to 6 carbon atoms which can be straight-chained or branched and havingat least 1 and particularly from 1 to 2 sites of alkynyl unsaturation.Particular non-limiting examples of alkynyl groups include acetylenic,ethynyl (—C≡CH), propargyl (—CH₂C≡CH), and the like.

“Alkoxy” refers to the group —OR where R is alkyl. Particular alkoxygroups include, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

“Alkoxycarbonyl” refers to a radical —C(O)-alkoxy where alkoxy is asdefined herein.

“Amino” refers to the radical —NH₂.

“Carboxyl” refers to the radical —C(O)OH.

“Dialkylamino” means a radical —NRR′ where R and R′ independentlyrepresent an alkyl, substituted alkyl, aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitutedcycloheteroalkyl, heteroaryl, or substituted heteroaryl group as definedherein.

“Halogen” or “halo” refers to chloro, bromo, fluoro or iodo.

“Hydroxy” refers to the radical —OH.

“Monoalkylamino” refers to the group alkyl-NR′—, wherein R′ is selectedfrom hydrogen and alkyl.

“Nitro” refers to the radical —NO₂.

“Thioalkoxy” refers to the group —SR where R is alkyl.

“Pharmaceutically acceptable salt” refers to any salt of a compound ofthis invention which retains its biological properties and which is nottoxic or otherwise undesirable for pharmaceutical use. Such salts may bederived from a variety of organic and inorganic counter-ions well knownin the art and include. Such salts include: (1) acid addition saltsformed with organic or inorganic acids such as hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic,trifluoroacetic, trichloroacetic, propionic, hexanoic,cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic,succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric,benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic,phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2)salts formed when an acidic proton present in the parent compound either(a) is replaced by a metal ion, e.g., an alkali metal ion, an alkalineearth ion or an aluminum ion, or alkali metal or alkaline earth metalhydroxides, such as sodium, potassium, calcium, magnesium, aluminum,lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with anorganic base, such as aliphatic, alicyclic, or aromatic organic amines,such as ammonia, methylamine, dimethylamine, diethylamine, picoline,ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylene-diamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, and the like.

Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium and the like, and whenthe compound contains a basic functionality, salts of non-toxic organicor inorganic acids, such as hydrohalides, e.g. hydrochloride andhydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate,trifluoroacetate, trichloroacetate, propionate, hexanoate,cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate,malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate,tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate,cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate),ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate,benzenesulfonate (besylate), 4-chlorobenzenesulfonate,2-naphthalenesulfonate, 4-toluenesulfonate, camphorate,camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate,glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate,lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate,salicylate, stearate, cyclohexylsulfamate, quinate, muconate and thelike.

The term “physiologically acceptable cation” refers to a non-toxic,physiologically acceptable cationic counterion of an acidic functionalgroup. Such cations are exemplified by sodium, potassium, calcium,magnesium, ammonium and tetraalkylammonium cations and the like.

“Solvate” refers to a compound of the present invention or a saltthereof, that further includes a stoichiometric or non-stoichiometricamount of solvent bound by non-covalent intermolecular forces. Where thesolvent is water, the solvate is a hydrate.

It is to be understood that compounds having the same molecular formulabut differing in the nature or sequence of bonding of their atoms or inthe arrangement of their atoms in space are termed “isomers.” Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers.”

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers”. When a compound has an asymmetriccenter, for example, when it is bonded to four different groups, a pairof enantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is designated (R) or(S) according to the rules of Cahn and Prelog (Cahn et al., 1966, Angew.Chem. 78:413-447, Angew. Chem., Int. Ed. Engl. 5:385-414: (errata:Angew. Chem., Int. Ed. Engl. 5:511); Prelog and Helmchen, 1982, Angew.Chem. 94:614-631, Angew. Chem. Internat. Ed. Eng. 21:567-583; Mata andLobo, 1993, Tetrahedron: Asymmetry 4:657-668) or can be characterized bythe manner in which the molecule rotates the plane of polarized lightand is designated dextrorotatory or levorotatory (i.e., as (+)- or(−)-isomers, respectively). A chiral compound can exist as eitherindividual enantiomer or as a mixture thereof. A mixture containingequal proportions of enantiomers is called a “racemic mixture.”

In certain embodiments, the compounds of this invention may possess oneor more asymmetric centers; such compounds can therefore be produced asthe individual (R)- or (S)-enantiomer or as a mixture thereof. Unlessindicated otherwise, for example by designation of stereochemistry atany position of a formula, the description or naming of a particularcompound in the specification and claims is intended to include bothindividual enantiomers and mixtures, racemic or otherwise, thereof.Methods for determination of stereochemistry and separation ofstereoisomers are well-known in the art. In particular embodiments, thepresent invention provides the stereoisomers of the compounds depictedherein upon treatment with base.

In certain embodiments, the compounds of the invention are“stereochemically pure.” A stereochemically pure compound or has a levelof stereochemical purity that would be recognized as “pure” by those ofskill in the art. Of course, this level of purity will be less than100%. In certain embodiments, “stereochemically pure” designates acompound that is substantially free of alternate isomers. In particularembodiments, the compound is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5% or 99.9% free of other isomers.

“Sarcosine” or “Sar” refers to the amino acid residue known to those ofskill in the art having the structure —N(Me)CH₂C(O)—. Those of skill inthe art might recognize sarcosine as N-methyl glycine.

As used herein, the terms “subject” and “patient” are usedinterchangeably herein. The terms “subject” and “subjects” refer to ananimal, such as a mammal including a non-primate (e.g., a cow, pig,horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey such as acynomolgous monkey, a chimpanzee and a human), and for example, a human.In one embodiment, the subject is refractory or non-responsive tocurrent treatments for hepatitis C infection. In another embodiment, thesubject is a farm animal (e.g., a horse, a cow, a pig, etc.) or a pet(e.g., a dog or a cat). In one embodiment, the subject is a human.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment or preventionof a disorder or one or more symptoms thereof. In certain embodiments,the term “therapeutic agent” refers to a compound of the invention. Incertain other embodiments, the term “therapeutic agent” refers does notrefer to a compound of the invention. In one embodiment, a therapeuticagent is an agent which is known to be useful for, or has been or iscurrently being used for the treatment or prevention of a disorder orone or more symptoms thereof.

“Therapeutically effective amount” means an amount of a compound orcomplex or composition that, when administered to a subject for treatinga disease, is sufficient to effect such treatment for the disease. A“therapeutically effective amount” can vary depending on, inter alia,the compound, the disease and its severity, and the age, weight, etc.,of the subject to be treated.

“Treating” or “treatment” of any disease or disorder refers, in oneembodiment, to ameliorating a disease or disorder that exists in asubject. In another embodiment, “treating” or “treatment” refers toameliorating at least one physical parameter, which may be indiscernibleby the subject. In yet another embodiment, “treating” or “treatment”refers to modulating the disease or disorder, either physically (e.g.,stabilization of a discernible symptom) or physiologically (e.g.,stabilization of a physical parameter) or both. In yet anotherembodiment, “treating” or “treatment” refers to delaying the onset ofthe disease or disorder.

As used herein, the terms “prophylactic agent” and “prophylactic agents”as used refer to any agent(s) which can be used in the prevention of adisorder or one or more symptoms thereof. In certain embodiments, theterm “prophylactic agent” refers to a compound of the invention. Incertain other embodiments, the term “prophylactic agent” does not refera compound of the invention. For example, a prophylactic agent is anagent which is known to be useful for, or has been or is currently beingused to the prevent or impede the onset, development, progression and/orseverity of a disorder.

As used herein, the terms “prevent,” “preventing” and “prevention” referto the prevention of the recurrence, onset, or development of one ormore symptoms of a disorder in a subject resulting from theadministration of a therapy (e.g., a prophylactic or therapeutic agent),or the administration of a combination of therapies (e.g., a combinationof prophylactic or therapeutic agents).

As used herein, the phrase “prophylactically effective amount” refers tothe amount of a therapy (e.g., prophylactic agent) which is sufficientto result in the prevention of the development, recurrence or onset ofone or more symptoms associated with a disorder (, or to enhance orimprove the prophylactic effect(s) of another therapy (e.g., anotherprophylactic agent).

As used herein, the term “in combination” refers to the use of more thanone therapies (e.g., one or more prophylactic and/or therapeuticagents). The use of the term “in combination” does not restrict theorder in which therapies (e.g., prophylactic and/or therapeutic agents)are administered to a subject with a disorder. A first therapy (e.g., aprophylactic or therapeutic agent such as a compound of the invention)can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes,45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapy (e.g., aprophylactic or therapeutic agent) to a subject with a disorder.

As used herein, the term “synergistic” refers to a combination of acompound of the invention and another therapy (e.g., a prophylactic ortherapeutic agent) which has been or is currently being used to prevent,manage or treat a disorder, which is more effective than the additiveeffects of the therapies. A synergistic effect of a combination oftherapies (e.g., a combination of prophylactic or therapeutic agents)permits the use of lower dosages of one or more of the therapies and/orless frequent administration of said therapies to a subject with adisorder. The ability to utilize lower dosages of a therapy (e.g., aprophylactic or therapeutic agent) and/or to administer said therapyless frequently reduces the toxicity associated with the administrationof said therapy to a subject without reducing the efficacy of saidtherapy in the prevention or treatment of a disorder). In addition, asynergistic effect can result in improved efficacy of agents in theprevention or treatment of a disorder. Finally, a synergistic effect ofa combination of therapies (e.g., a combination of prophylactic ortherapeutic agents) may avoid or reduce adverse or unwanted side effectsassociated with the use of either therapy alone.

The term “label” refers to a display of written, printed or graphicmatter upon the immediate container of an article, for example thewritten material displayed on a vial containing a pharmaceuticallyactive agent.

The term “labeling” refers to all labels and other written, printed orgraphic matter upon any article or any of its containers or wrappers oraccompanying such article, for example, a package insert orinstructional videotapes or DVDs accompanying or associated with acontainer of a pharmaceutically active agent.

5.2 Embodiments of the Invention

The present invention is based, in part, on the discovery that compoundsof the invention are effective for the treatment and prevention ofhepatitis C infection in a subject in need thereof. Accordingly, thepresent invention provides methods of treating hepatitis C infection ina subject in need thereof. The present invention further providesmethods of preventing hepatitis C infection in a subject in needthereof. In general, the methods of the invention comprise the step ofadministering to the subject in need thereof an amount of a compound ofthe invention effective for the treatment or prevention of the hepatitisC infection in combination with a second agent effective for thetreatment or prevention of the infection. Methods of treatment aredescribed in detail in the sections below. The compound can be anycompound of the invention as described in the sections below, and thesecond agent can be any second agent described in the sections below. Incertain embodiments, the compound is in the form of a pharmaceuticalcomposition or dosage form, as described in the sections below.

While not intending to be bound by any particular theory of operation,it is believed that compounds of the invention inhibit hepatitis C virus(HCV) replication by a mechanism distinct from that of current HCVtherapy. Current therapy for HCV, as mentioned above, isco-administration of interferon and ribavirin. It is believed that thecurrent therapy operates by modulation of the immune system of a subjectto treat or prevent infection by HCV. It is believed that compounds ofthe present invention operate by modulating or inhibiting cellularprocesses critical for HCV replication in a host. Such mechanisms arediscussed in the examples below. Operating by a novel mechanism, thecompounds, compositions and methods of the invention offer a noveltherapy for the treatment or prevention of HCV infection. As such theyare advantageous for any subject infected with, or at risk for infectionwith, HCV and particularly for subjects that have not responded tocurrent therapy.

In embodiments of the invention, the subject can be any subject infectedwith, or at risk for infection with, HCV. Infection or risk forinfection can be determined according to any technique deemed suitableby the practitioner of skill in the art. In one embodiment, subjects arehumans infected with HCV.

The HCV can be any HCV known to those of skill in the art. There are atleast six genotypes and at least 50 subtypes of HCV currently known tothose of skill in the art. The HCV can be of any genotype or subtypeknown to those of skill. In certain embodiments, the HCV is of agenotype or subtype not yet characterized. In certain embodiments, thesubject is infected with HCV of a single genotype. In certainembodiments, the subject is infected with HCV of multiple subtypes,quasispecies, or multiple genotypes.

In certain embodiments, the HCV is genotype 1 and can be of any subtype.For instance, in certain embodiments, the HCV is subtype 1a, 1b or 1c.It is believed that HCV infection of genotype 1 responds poorly tocurrent interferon therapy. Methods of the present invention can beadvantageous for therapy of HCV infection with genotype 1.

In certain embodiments, the HCV is other than genotype 1. In certainembodiments, the HCV is genotype 2 and can be of any subtype. Forinstance, in certain embodiments, the HCV is subtype 2a, 2b or 2c. Incertain embodiments, the HCV is genotype 3 and can be of any subtype.For instance, in certain embodiments, the HCV is subtype 3a, 3b or 10a.In certain embodiments, the HCV is genotype 4 and can be of any subtype.For instance, in certain embodiments, the HCV is subtype 4a. In certainembodiments, the HCV is genotype 5 and can be of any subtype. Forinstance, in certain embodiments, the HCV is subtype 5a. In certainembodiments, the HCV is genotype 6 and can be of any subtype. Forinstance, in certain embodiments, the HCV is subtype 6a, 6b, 7b, 8b, 9aor 11a. See, e.g., Simmonds, 2004, J Gen Virol. 85:3173-88; Simmonds,2001, J. Gen. Virol., 82, 693-712, the contents of which areincorporated by reference in their entirety.

In certain embodiments of the invention, the subject has never receivedtherapy or prophylaxis for HCV infection. In further embodiments of theinvention, the subject has previously received therapy or prophylaxisfor HCV infection. For instance, in certain embodiments, the subject hasnot responded to HCV therapy. Indeed, under current interferon therapy,up to 50% or more HCV subjects do not respond to therapy. In certainembodiments, the subject can be a subject that received therapy butcontinued to suffer from viral infection or one or more symptomsthereof. In certain embodiments, the subject can be a subject thatreceived therapy but failed to achieve a sustained virologic response.In certain embodiments, the subject has received therapy for HCVinfection but has failed show a 2 log₁₀ decline in HCV RNA levels after12 weeks of therapy. It is believed that subjects who have not shownmore than 2 log₁₀ reduction in serum HCV RNA after 12 weeks of therapyhave a 97-100% chance of not responding. Since the compounds of thepresent invention act by mechanism other than current HCV therapy, it isbelieved that compounds of the invention should be effective in treatingsuch nonresponders.

In certain embodiments, the subject is a subject that discontinued HCVtherapy because of one or more adverse events associated with thetherapy. In certain embodiments, the subject is a subject where currenttherapy is not indicated. For instance, certain therapies for HCV areassociated with neuropsychiatric events. Interferon (IFN)-alfa plusribavirin is associated with a high rate of depression. Depressivesymptoms have been linked to a worse outcome in a number of medicaldisorders. Life-threatening or fatal neuropsychiatric events, includingsuicide, suicidal and homicidal ideation, depression, relapse of drugaddiction/overdose, and aggressive behavior have occurred in subjectswith and without a previous psychiatric disorder during HCV therapy.Interferon-induced depression is a limitation for the treatment ofchronic hepatitis C, especially for subjects with psychiatric disorders.Psychiatric side effects are common with interferon therapy andresponsible for about 10% to 20% of discontinuations of current therapyfor HCV infection.

Accordingly, the present invention provides methods of treating orpreventing HCV infection in subjects where the risk of neuropsychiatricevents, such as depression, contraindicates treatment with current HCVtherapy. The present invention also provides methods of treating orpreventing HCV infection in subjects where a neuropsychiatric event,such as depression, or risk of such indicates discontinuation oftreatment with current HCV therapy. The present invention furtherprovides methods of treating or preventing HCV infection in subjectswhere a neuropsychiatric event, such as depression, or risk of suchindicates dose reduction of current HCV therapy.

Current therapy is also contraindicated in subjects that arehypersensitive to interferon or ribavirin, or both, or any othercomponent of a pharmaceutical product for administration of interferonor ribavirin. Current therapy is not indicated in subjects withhemoglobinopathies (e.g., thalassemia major, sickle-cell anemia) andother subjects at risk from the hematologic side effects of currenttherapy. Common hematologic side effects include bone marrowsuppression, neutropenia and thrombocytopenia. Furthermore, ribavirin istoxic to red blood cells and is associated with hemolysis. Accordingly,the present invention also provides methods of treating or preventingHCV infection in subjects hypersensitive to interferon or ribavirin, orboth, subjects with a hemoglobinopathy, for instance thalassemia majorsubjects and sickle-cell anemia subjects, and other subjects at riskfrom the hematologic side effects of current therapy.

In certain embodiments, the subject has received HCV therapy anddiscontinued that therapy prior to administration of a method of theinvention. In further embodiments, the subject has received therapy andcontinues to receive that therapy along with administration of a methodof the invention. The methods of the invention can be co-administeredwith other therapy for HCV according to the judgment of one of skill inthe art. In certain embodiments, the methods or compositions of theinvention can be co-administered with a reduced dose of the othertherapy for HCV.

In certain embodiments, the present invention provides methods oftreating a subject that is refractory to treatment with interferon. Forinstance, in some embodiments, the subject can be a subject that hasfailed to respond to treatment with one or more agents selected from thegroup consisting of interferon, interferon α, pegylated interferon α,interferon plus ribavirin, interferon α plus ribavirin and pegylatedinterferon α plus ribavirin. In some embodiments, the subject can be asubject that has responded poorly to treatment with one or more agentsselected from the group consisting of interferon, interferon α,pegylated interferon α, interferon plus ribavirin, interferon α plusribavirin and pegylated interferon α plus ribavirin. A pro-drug form ofribavirin, such as taribavirin, may also be used.

In further embodiments, the present invention provides methods oftreating HCV infection in subjects that are pregnant or might getpregnant since current therapy is also contraindicated in pregnantwomen.

In certain embodiments, the subject has, or is at risk for, co-infectionof HCV with HIV. For instance, in the United States, 30% of HIV subjectsare co-infected with HCV and evidence indicates that people infectedwith HIV have a much more rapid course of their hepatitis C infection.Maier and Wu, 2002, World J Gastroenterol 8:577-57. The methods of theinvention can be used to treat or prevent HCV infection in suchsubjects. It is believed that elimination of HCV in these subjects willlower mortality due to end-stage liver disease. Indeed, the risk ofprogressive liver disease is higher in subjects with severeAIDS-defining immunodeficiency than in those without. See, e.g., Lesenset al., 1999, J Infect Dis 179:1254-1258. In one embodiment, compoundsof the invention have been shown to suppress HIV in HIV subjects. See,e.g., U.S. Pat. Nos. 5,977,067; 5,994,299, 5,948,884 and 6,583,265 andPCT publication nos. WO99/32512, WO99/67280, the contents of which arehereby incorporated by reference in their entireties. Thus, in certainembodiments, the present invention provides methods of treating orpreventing HIV infection and HCV infection in subjects in need thereof.

In certain embodiments, the methods or compositions of the invention areadministered to a subject following liver transplant. Hepatitis C is aleading cause of liver transplantation in the U.S., and many subjectsthat undergo liver transplantation remain HCV positive followingtransplantation. The present invention provides methods of treating suchrecurrent HCV subjects with a compound or composition of the invention.In certain embodiments, the present invention provides methods oftreating a subject before, during or following liver transplant toprevent recurrent HCV infection.

5.2.1 Compounds Used in the Invention

In certain embodiments, the compound of the invention is a cyclosporinderivative effective for the treatment or prevention of hepatitis Cinfection in a subject in need thereof. Unless noted otherwise, the term“cyclosporin” as used herein refers to the compound cyclosporin A asknown to those of skill in the art. See, e.g., Ruegger et al., 1976,Helv. Chim. Acta. 59:1075-92; Borel et al., 1977, Immunology 32:1017-25;the contents of which are hereby incorporated by reference in theirentireties. The term “cyclosporin derivative” refers to any cyclosporinderivative with activity against hepatitis C infection, whether thederivative is natural, synthetic or semi-synthetic.

In particular embodiments, the cyclosporin derivative differs fromcyclosporin A at the third position, i.e. the N-methyl glycine position,known to those of skill in the art. In certain embodiments, thecyclosporin derivative is a 3-ether cyclosporin. In further embodiments,the cyclosporin derivative is a 3-thioether cyclosporin. The cyclosporinderivative can further comprise other cyclosporin modifications known tothose of skill in the art. In further embodiments, the cyclosporinfurther comprises a 4-gamma-hydroxymethylleucine residue. Accordingly,in certain embodiments, the cyclosporin derivative is a 3-ether,4-gamma-hydroxymethylleucine. In further embodiments, the cyclosporinderivative is a 3-thioether, 4-gamma-hydroxymethylleucine.

In certain embodiments, the present invention provides methods oftreating or preventing hepatitis C infection in a subject comprisingadministering to the subject a therapeutically or prophylacticallyeffective amount of a cyclosporin derivative of general formula (I), ora pharmaceutically acceptable salt or solvate thereof:

along with a second agent effective for treating or preventing HCVinfection.

In formula (I), A, B, X, R¹ and R² are as defined above.

In certain embodiments, A is according to formula (IIa). In furtherembodiments, A is according to formula (IIb). In one embodiment, A is aresidue of formula (IIa) above.

In certain embodiments, B is ethyl.

In certain embodiments, R¹ is 2-aminoethyl, 2-aminopropyl,2-monoalkylaminoethyl, 2-monoalkylaminopropyl; 2-dialkylaminoethyl2-dialkylaminopropyl, 2-monocycloalkylaminoethyl,2-monocycloalkylaminopropyl, 2-dicycloalkylaminoethyl or2-dialkylaminopropyl wherein alkyl is straight- or branched-chaincontaining from one to four carbon atoms, and cycloalkyl contains fromthree to six carbon atoms.

In a further embodiment, R¹ is straight- or branched-chain alkylcontaining from one to four carbon atoms, in another embodiment, one ortwo carbon atoms, optionally substituted by one group R³.

In certain embodiments, R² is isobutyl. In other embodiments, R² is2-hydroxyisobutyl.

In one embodiment, X is oxygen or sulfur. In certain embodiments, X isoxygen. In further embodiments, X is sulfur.

In certain embodiments R³ is selected from the group consisting ofhalogen, hydroxy, carboxyl, alkoxycarbonyl, —NR⁴R⁵ and—NR⁶(CH₂)_(m)NR⁴R⁵.

R³ is hydroxy or —NR⁴R⁵, wherein R⁴ and R⁵, which may be the same ordifferent, each represent hydrogen or straight- or branched-chain alkylcontaining from one to six carbon atoms or from one to four carbonatoms. In a further embodiment, R³ is —NR⁴R⁵.

In certain embodiments, when X is sulfur, R¹ is selected from the groupconsisting of N,N-dimethylaminoethyl, N,N-diethylaminoethyl,N-methyl-N-tert-butylaminoethyl and N-ethyl-N-tert-butylaminoethyl.

In certain embodiments, X is sulfur, R² is isobutyl and R¹ is selectedfrom the group consisting of N,N-dimethylaminoethyl,N,N-diethylaminoethyl, N-methyl-N-tert-butylaminoethyl andN-ethyl-N-tert-butylaminoethyl.

In certain embodiments, X is sulfur, R² is 2-hydroxyisobutyl and R¹ isselected from the group consisting of N,N-dimethylaminoethyl,N,N-diethylaminoethyl, N-methyl-N-tert-butylaminoethyl andN-ethyl-N-tert-butylaminoethyl.

Further compounds of formula (I) are those in which R¹ is straight- orbranched chain alkyl containing from two to six carbon atoms optionallysubstituted by a group R³; or straight- or branched chain alkenylcontaining from two to four carbon atoms; and R³ is hydroxy, —NR⁴R⁵ ormethoxy.

Further compounds of formula (I) are those in which each of R⁴ and R⁵,which may be the same or different, is hydrogen; straight- orbranched-chain alkyl comprising from one to four carbon atoms, or R⁴ andR⁵, together with the nitrogen atom to which they are attached, form asaturated ring containing six ring atoms; the ring atoms other than thenitrogen atom being independently selected from carbon and oxygen.

In a further embodiment, R³ is selected from the group consisting ofhalogen, hydroxy, carboxyl, alkoxycarbonyl, —NR⁴R⁵ and—NR⁶(CH₂)_(m)NR⁴R⁵. The variable m can be an integer from two to four.

In certain embodiments, halogen is fluoro, chloro or bromo. In oneembodiment, halogen is fluoro or chloro.

In one embodiment, compounds of formula (I) in which X is oxygen and R¹is 2-methoxyethyl, or pharmaceutically acceptable salts thereof are usedin the methods and compositions provided herein.

In another embodiment, compounds of formula (I) in which X is oxygen orsulfur and R¹ is propyl substituted by —NR⁴R⁵ or methoxy, orpharmaceutically acceptable salts thereof are used in the methods andcompositions provided herein.

Among the compounds used in the methods and compositions of the presentinvention, are the cyclosporin derivatives listed below:

-   3-methoxycyclosporin;-   3-ethoxycyclosporin;-   3-propoxycyclosporin;-   3-isopropoxycyclosporin;-   3-(2-aminoethoxy)cyclosporin;-   3-(2-N-methylaminoethoxy)cyclosporin;-   3-(2-N-ethylaminoethoxy)cyclosporin;-   3-(2-dimethylaminoethoxy)cyclosporin;-   3-(2-diethylaminoethoxy)cyclosporin;-   3-(2-tert-butyl-methylaminoethoxy)cyclosporin;-   3-(2-tert-butyl-ethylaminoethoxy)cyclosporin;-   3-[(R)-2-(N,N-dimethylamino)ethylthio-Sar]-4-[4′-hydroxy-MeLeu]-cyclosporin;-   3-[(R)-2-(N,N-dimethylamino)ethylthio-Sar]-cyclosporin;-   3-[(R)-2-(hydroxy)ethylthio-Sar]-4-[4′-hydroxy-MeLeu]-cyclosporin;-   3-[(R)-2-(hydroxy)ethylthio-Sar]-cyclosporin;-   3-[(R)-2-(N,N-diethylamino)ethylthio-Sar]-4-[4′-hydroxy-MeLeu]-cyclosporin;-   3-[(R)-2-(N,N-diethylamino)ethylthio-Sar]-cyclosporin;-   3-(sec-butoxy)cyclosporin;-   3-[2-(1,4-dihydropyrid-1-yl)ethoxy)cyclosporin;    and their pharmaceutically acceptable salts.

In certain embodiments, compounds useful in the methods and compositionsof the invention include the following: Compound Name A3-methoxycyclosporin B 3-(2-aminoethoxy)cyclosporin C3-(2-N,N-dimethylaminoethoxy)cyclosporin D 3-(isopropoxy)cyclosporin E3-(2-ethylbutoxy)cyclosporin F 3-(2,2-dimethylpropoxy)cyclosporin G3-(2-hydroxyethoxy)cyclosporin H 3-(3-hydroxypropoxy)cyclosporin I3-[2-(N-methylamino)ethoxy]cyclosporin J3-[2-(N-methyl-N-isopropylamino)ethoxy]cyclosporin K3-[2-(piperidin-1-yl)ethoxy]cyclosporin L3-[2-(N-morpholine)ethoxy)cyclosporin M 3-ethoxycyclosporin N3-(2-methoxyethylthio)-4- (gamma-hydroxymethylleucine)cyclosporin O3-[(R)-2-(N,N-dimethylamino)ethylthio-Sar]-4-(gamma-hydroxymethylleucine)cyclosporin P 3-ethylthiocyclosporin Q3-propenylthiocyclosporin R 3-[(2-methoxy)ethylthio]cyclosporin S3-(methylthio)-4-(gamma- hydroxymethylleucine)cyclosporin T3-(methoxy)-4-(gamma-hydroxymethylleucine)cyclosporin U3-(prop-2-ene-1-oxy)-4-(gamma- hydroxymethylleucine)cyclosporin V3-(isopropoxy)-4-(gamma- hydroxymethylleucine)cyclosporin W3-(ethoxy)-4-(gamma-hydroxymethylleucine)cyclosporin X3-[2-(methoxy)ethoxy]-4- (gamma-hydroxymethylleucine)cyclosporin Y3-[3-(methoxy)propoxy]-4- (gamma-hydroxymethylleucine)cyclosporin

In particular embodiments, the present invention provides a method oftreating or preventing hepatitis C virus infection in a subject byadministering, to a subject in need thereof, an effective amount of acompound of the invention selected from the group consisting ofcompounds A to Y, or a pharmaceutically acceptable salt thereof, and aneffective amount of a second agent as described below.

In one embodiment, the compound is Compound O, or a pharmaceuticallyacceptable salt thereof, due to its high level of activity and itstoxicological profile, as described in the examples below.

In another embodiment, the methods and compositions provided herein useCompounds C and T, or pharmaceutically acceptable salts thereof.

In certain embodiments, cyclosporin derivatives according to theinvention in which R¹ is alkyl substituted by one or more groups R³,where R³ is —NR⁴R⁵ or —NR⁶(CH₂)_(m)NR⁴R⁵ and R⁴, R⁵ and R⁶ are asdefined above, can be converted into addition salts with acids by knownmethods. It is understood that these salts also come within the scope ofthe present invention. Exemplary salts of the invention, and methods oftheir preparation, are described in the sections below.

Mention may be made, as examples of pharmaceutically acceptable salts,of the salts with alkali metals, e.g., sodium, potassium or lithium, orwith alkaline-earth metals, e.g., magnesium or calcium, the ammoniumsalt or the salts of nitrogenous bases, e.g., ethanolamine,diethanolamine, trimethylamine, triethylamine, methylamine, propylamine,diisopropylamine, N,N-dimethylethanolamine, benzylamine,dicyclohexylamine, N-benzylphenethylamine, N,N′-dibenzylethylenediamine,diphenylenediamine, benzhydrylamine, quinine, choline, arginine, lysine,leucine or dibenzylamine.

Mention may be made, as examples of addition salts with pharmaceuticallyacceptable acids, of the salts formed with inorganic acids, e.g.,hydrochlorides, hydrobromides, sulfates, nitrates or phosphates, or withorganic acids, e.g., succinates, fumarates, tartrates, acetates,propionates, maleates, citrates, methanesulfonates, ethanesulfonates,p-toluenesulfonates, isethionates or embonates, or with substitutionderivatives of these compounds. Preferred salts are succinate,phosphate, citrate, acetate, hydrochlorides, methanesulfonate andpropionate. Certain of these salts are novel and as such constitute afurther feature of the present invention.

In useful embodiments of the invention, the compound is in a pure form.Purity can be any purity known to those of skill in the art such asabsolute purity, stereochemical purity or both. In certain embodiments,the compound of the invention is at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% pure. In certain embodiments, thecompound of the invention is at least 90% pure. In further embodiments,of the invention, the compound is at least 98% pure. Methods ofpurifying compounds of the invention are described below.

5.2.2 Preparation of Compounds of the Invention

The compounds of the invention can be prepared, isolated or obtained byany method apparent to those of skill in the art. Exemplary methods ofpreparation are described in detail in the examples below.

In addition, cyclosporins substituted in the 3-position by thioether orether groups can be prepared according to methods described in U.S. Pat.Nos. 5,977,067; 5,994,299, 5,948,884 and 6,583,265 and in PCTpublication nos. WO99/32512, WO99/67280. The contents of thesereferences are hereby incorporated by reference in their entireties.

Compounds can be purified after synthesis by any technique apparent tothose of skill in the art for purifying cyclosporin derivatives. Incertain embodiments, a compound of the invention is purified bychromatography. For instance, a compound of the invention can bepurified using high-performance liquid chromatography (HPLC). An usefulexample of the HPLC purification is as follows: An HPLC column ofdimensions 10 mm (d)×50 mm (1) containing a 5-μm reverse-phasestationary phase (octadecyl-silane or octa-silane) is equilibrated witha mobile phase comprising 0.1% formic acid, 50 to 90% water, and 50 to10% acetonitrile. Importantly, the column is heated to at least 65° C.,or potentially up to 85° C. The mobile phase flows at 10 to 16 mL/minuteand is heated to 60° C. Approximately 5 to 25 mg of a cyclosporinderivative is loaded on the column in 0.1 to 0.8 mL of a solvent,preferably dimethylsulfoxide. The mobile phase flow is maintained, andits composition is adjusted in a linear gradient up to 90% or 100%acetonitrile over a period of 20 to 60 minutes. Compound peaks aredetected using evaporative light scattering detection and/orvariable-ultraviolet detection at a wavelength setting of 205 to 215 nm.Compound peaks are collected in the mobile phase which is removed invacuo; samples are thoroughly dried in vacuo and analyzed by NMR, IR,and HPLC-MS to determine identity and purity.

5.2.3 Pharmaceutical Salts of Compounds of the Invention

As discussed above, a cyclosporin derivative of the invention can be ina neutral form, or in a salt form. The salt form can be any salt formknown to those of skill in the art. Particularly useful salt forms arethose that are coordinated with phosphate, citrate, acetate, chloride,methanesulfonate or propionate.

Where a compound of the present invention, e.g. a compound of theinvention, is substituted with a basic moiety, an acid addition salt canbe formed. The acid which can be used to prepare an acid addition saltincludes preferably that which produces, when combined with the freebase, a pharmaceutically acceptable salt, that is, a salt whose anion isnon-toxic to a subject in the pharmaceutical doses of the salt.Pharmaceutically acceptable salts within the scope of the invention arethose derived from the following acids: mineral acids such ashyrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,sulfamic acid and nitric acid; and organic acids such as acetic,trifluoroacetic, trichloroacetic, propionic, hexanoic,cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic,succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric,benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic,phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids.

The corresponding acid addition salts include hydrohalides, e.g.hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate,acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate,cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate,malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate,tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate,cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate),ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate,benzenesulfonate (besylate), 4-chlorobenzenesulfonate,2-naphthalenesulfonate, 4-toluenesulfonate, camphorate,camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate,glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate,lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate,salicylate, stearate, cyclohexylsulfamate, quinate, muconate and thelike.

According to a further feature of the invention, acid addition salts ofthe compounds of this invention can be prepared by reaction of the freebase with the appropriate acid, by the application or adaptation ofknown methods. For example, the acid addition salts of the compounds ofthis invention can be prepared either by dissolving the free base inaqueous or aqueous-alcohol solution or other suitable solventscontaining the appropriate acid and isolating the salt by evaporatingthe solution, or by reacting the free base and acid in an organicsolvent, in which case the salt separates directly or can be obtained byconcentration of the solution.

The acid addition salts of the compounds of this invention, e.g.compounds of the invention, can be regenerated from the salts by theapplication or adaptation of known methods. For example, parentcompounds of the invention can be regenerated from their acid additionsalts by treatment with an alkali, e.g., aqueous sodium bicarbonatesolution or aqueous ammonia solution.

Where a compound of the invention, e.g. a compound of the invention, issubstituted with an acid moiety, base addition salts can be formed.Pharmaceutically acceptable salts, including for example alkali andalkaline earth metal salts, within the scope of the invention are thosederived from the following bases: sodium hydride, sodium hydroxide,potassium hydroxide, calcium hydroxide, magnesium hydroxide, aluminumhydroxide, lithium hydroxide, zinc hydroxide, barium hydroxide, andorganic amines such as aliphatic, alicyclic, or aromatic organic amines,such as ammonia, methylamine, dimethylamine, diethylamine, picoline,ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylene-diamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, and the like.

Metal salts of compounds of the present invention, e.g. compounds of theinvention, can be obtained by contacting a hydride, hydroxide, carbonateor similar reactive compound of the chosen metal in an aqueous ororganic solvent with the free acid form of the compound. The aqueoussolvent employed may be water or it may be a mixture of water with anorganic solvent, for example, an alcohol such as methanol or ethanol, aketone such as acetone, an aliphatic ether such as tetrahydrofuran, oran ester such as ethyl acetate. Such reactions are normally conducted atambient temperature but they may, if desired, be conducted with heating.

Amine salts of compounds of the present invention, e.g. compounds of theinvention, can be obtained by contacting an amine in an aqueous ororganic solvent with the free acid form of the compound. Suitableaqueous solvents include water and mixtures of water with alcohols suchas methanol or ethanol, ethers such as tetrahydrofuran, nitriles, suchas acetonitrile, or ketones such as acetone. Amino acid salts may besimilarly prepared.

The base addition salts of the compounds of this invention, e.g.compounds of the invention, can be regenerated from the salts by theapplication or adaptation of known methods. For example, parentcompounds of the invention can be regenerated from their base additionsalts by treatment with an acid, e.g., hydrochloric acid.

5.3 Second Agents Useful in the Methods of the Invention

The present invention provides methods of treatment of prevention thatcomprise the administration of a second agent effective for thetreatment or prevention of HCV infection in a subject in need thereof.The second agent can be any agent known to those of skill in the art tobe effective for the treatment or prevention of the HCV infection. Thesecond agent can be a second agent presently known to those of skill inthe art, or the second agent can be second agent later developed for thetreatment or prevention of HCV. In certain embodiments, the second agentis presently approved for the treatment or prevention of HCV.

In certain embodiments, a compound of the invention is administered incombination with one second agent. In further embodiments, a secondagent is administered in combination with two second agents. In stillfurther embodiments, a second agent is administered in combination withtwo or more second agents.

Suitable second agents include small-molecule, orally bioavailableinhibitors of the HCV enzymes, nucleic-acid-based agents that attackviral RNA, agents that can modulate the host immune response. Exemplarysecond agents include: (i) current approved therapies (peg-interferonplus ribavirin), (ii) HCV-enzyme targeted compounds, (iii)viral-genome-targeted therapies (e.g., RNA interference or RNAi), and(iv) immunomodulatory agents such as ribavirin, interferon (INF) andToll-receptor agonists.

In certain embodiments, the second agent is a modulator of the NS3-4Aprotease. The NS3-4A protease is a heterodimeric protease, comprisingthe amino-terminal domain of the NS3 protein and the small NS4Acofactor. Its activity is essential for the generation of components ofthe viral RNA replication complex.

One useful NS3-4A protease inhibitor is BILN 2061 (Ciluprevir;Boehringer Ingelheim), a macrocyclic mimic of peptide productinhibitors. Although clinical trials with BILN 2061 were halted(preclinical cardiotoxicity), it was the first NS3 inhibitor to betested in humans. See Lamarre et al., 2003, Nature 426:186-189, thecontents of which are hereby incorporated by reference in theirentirety.

Another useful NS3-4A protease inhibitor is VX-950 (Vertex/Mitsubishi),a protease-cleavage-product-derived peptidomimetic inhibitor of theNS3-4A protease. It is believed to be stabilized into the enzyme'sactive site through a ketoamide. See, e.g. Lin et al., 2005, J BiolChem., Vol. 280(44) pp. 36784-36791, 2005, manuscript M506462200(epublication); Summa, 2005, Curr Opin Investig Drugs. 6:831-7, thecontents of which are hereby incorporated by reference in theirentireties.

Further useful NS3-4A protease inhibitor that are ketoamide inhibitorsinclude those disclosed in U.S. Ser. No. 09/908,955, published as US2004/0254117, the contents of which are hereby incorporated by referencein their entirety. Among the compounds disclosed therein is (1R, 2S,5S)-3-azabicyclo[3,1,0]hexane-2-carboxamide,N-[3-amino-1-cyclobutylmethyl)-2,3-dioxopropyl]-3-[(2S)-2-[[[1,1-dimethylethyl]amino]carbonylamino]-3,3-dimethyl-1-oxobutyl]-6,6-dimethyl,known as SCH503034. Another ketoamide inhibitor of NS3-4A protease is1,1-dimethylethyl[1(S)-[[(1R,5S)-2(S)-[[[1-(cyclopropylmethyl)-3-[[2-[[2-(dimethylamino)-2-oxo-1(S)-phenylethyl]-amino]-2-oxoethyl]amino]-2,3-dioxopropyl]amino]carbonyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]carbonyl]-2,2-dimethylpropyl]carbamate,known as SCH6 (also known as SCH 446211).

In certain embodiments, the second agent is a modulator of the HCV NS5BThe RNA-dependent RNA polymerase (RdRp). Contained within the NS5Bprotein, RdRp synthesizes RNA using an RNA template. This biochemicalactivity is not present in mammalian cells.

One useful modulator of RdRp is NM283 (Valopicitabine; Idenix/Novartis).NM283, is an oral prodrug (valine ester) of NM107 (2′-C-methyl-cytidine)in phase II trials for the treatment or prevention of HCV infection.See, e.g., U.S. Patent Application Publication No. 20040077587, thecontents of which are hereby incorporated by reference in theirentirety.

Other useful modulators of RdRp include 7-deaza nucleoside analogs. Forinstance, 7-Deaza-2′-C-methyl-adenosine is a potent and selectiveinhibitor of hepatitis C virus replication with excellentpharmacokinetic properties. Olsen et al., 2004, Antimicrob. AgentsChemother. 48:3944-3953, the contents of which are hereby incorporatedby reference in their entirety.

In further embodiments, the second agent is a non-nucleoside modulatorof NS5B. At least three different classes of non-nucleoside inhibitors(NNI) of NS5B inhibitors are being evaluated in the clinic.

Useful non-nucleoside modulators of NS5B include JTK-003 and JTK-009.JTK-003 has been advanced to phase II. Useful non-nucleoside modulatorsof NS5B include the 6,5-fused heterocyclic compounds based on abenzimidazole or indole core. See, e.g., Hashimoto et al., WO 00147883,the contents of which are hereby incorporated by reference in theirentirety.

Further useful polymerase NNIs include R803 (Rigel) and HCV-371, HCV-086and HCV-796 (ViroPharma/Wyeth). Additional useful NNIs include thiophenederivatives that are reversible allosteric inhibitors of the NS5Bpolymerase and bind to a site that is close to, but distinct from, thesite occupied by benzimidazole-based inhibitors. See, e.g., Biswal, etal., 2005, J. Biol. Chem. 280, 18202-18210 (2005).

Further useful NNIs for the methods of the invention includebenzothiadiazines, such as benzo-1,2,4-thiadiazines. Derivatives ofbenzo-1,2,4-thiadiazine have been shown to be highly selectiveinhibitors of the HCV RNA polymerase. Dhanak, et al., 2002, J. Biol.Chem. 277:38322-38327, the contents of which are hereby incorporated byreference in their entirety.

Further useful NNIs for the methods of the invention, and theirmechanisms, are described in LaPlante et al., 2004 Angew Chem. Int. Ed.Engl. 43:4306-4311; Tomei et al., 2003, J. Virol. 77:13225-13231; DiMarco et al., 2005, J. Biol. Chem. 280:29765-70; Lu, H., WO 2005/000308;Chan et al., 2004, Bioorg. Med. Chem. Lett. 14:797-800; Chan et al.,2004, Bioorg. Med. Chem. Lett. 14:793-796; Wang et al., 2003, J. Biol.Chem. 278:9489-9495; Love, et al., 2003, J. Virol. 77:7575-7581; Gu etal., 2003, J. Biol. Chem. 278:16602-16607; Tomei et al., 2004, J. Virol.78:938-946; and Nguyen et al., 2003, Antimicrob. Agents Chemother.47:3525-3530; the contents of each are hereby incorporated by referencein their entireties.

In a further embodiment, the second agent is an agent that is capable ofinterfering with HCV RNA such as small inhibitory RNA (siRNA) or a shorthairpin RNA (shRNA) directed to an HCV polynucleotide. In tissueculture, siRNA and vector-encoded short hairpin RNA shRNA directedagainst the viral genome, effectively block the replication of HCVreplicons. See, e.g., Randall et al., 2003, Proc. Natl. Acad. Sci. USA100:235-240, the contents of which are hereby incorporated by referencein their entirety.

In a further embodiment, the second agent is an agent that modulates thesubject's immune response. For instance, in certain embodiments, thesecond agent can be a presently approved therapy for HCV infection suchas an interferon (IFN), a pegylated IFN, an IFN plus ribavirin or apegylated IFN plus ribavirin. In one embodiment, interferons includeIFNα, IFNα2a and IFNα2b, and particularly pegylated IFNα2a (PEGASYS®) Orpegylated IFNα2b (PEG-INTRON®). In certain embodiments the second agentcan be a reversible inhibitor of inosine monophosphate dehydrogenase(IMPDH), such as mycophenloic acid or a derivative thereof e.g.mycophenylate mofetil (CELLCEPT®). Another example of an IMPDH inhibitoris merimepodib,[(3S)-oxolan-3-yl]N-[[3-[[3-methoxy-4-(1,3-oxazol-5-yl)phenyl]carbamoylamino]phenyl]methyl]carbamate(known as VX-497).

In a further embodiment, the second agent is a modulator of a Toll-likereceptors (TLR). It is believed that TLRs are targets for stimulatinginnate anti-viral response. Suitable TLRs include, bur are not limitedto, TLR3, TLR7, TLR8 and TLR9. It is believed that toll-like receptorssense the presence of invading microorganisms such as bacteria, virusesand parasites. They are expressed by immune cells, includingmacrophages, monocytes, dendritic cells and B cells. Stimulation oractivation of TLRs can initiate acute inflammatory responses byinduction of antimicrobial genes and pro-inflammatory cytokines andchemokines.

In certain embodiments, the second agent is a polynucleotide comprisinga CpG motif. Synthetic oligonucleotides containing unmethylated CpGmotifs are potent agonists of TLR-9. Stimulation of dendritic cells withthese oligonucleotides results in the production of tumour necrosisfactor-alpha, interleukin-12 and IFN-alpha. TLR-9 ligands are alsopotent stimulators of B-cell proliferation and antibody secretion. Oneuseful CpG-containing oligonucleotide is CPG-10101 (Actilon; ColeyPharmaceutical Group) which has been evaluated in the clinic.

Another useful modulator of a TLR is ANA975 (Anadys). ANA975 is believedto act through TLR-7, and is known to elicit a powerful anti-viralresponse via induction and the release of inflammatory cytokines such asIFN-alpha.

In another embodiment, the second agent is Celgosivir. Celgosivir is analpha-glucosidase I inhibitor and acts through host-directedglycosylation. In preclinical studies, celgosivir has demonstratedstrong synergy with IFNα plus ribavirin. See, e.g., Whitby et al., 2004,Antivir Chem Chemother. 15(3):141-51. Celgosivir is currently beingevaluated in a Phase II monotherapy study in chronic HCV patients inCanada.

Further immunomodulatory agents, and their mechanisms or targets, aredescribed in Schetter & Vollmer, 2004, Curr. Opin. Drug Discov. Dev.7:204-210; Takeda et al., 2003, Annu. Rev. Immunol. 21:335-376; Lee etal., 2003, Proc. Natl. Acad. Sci. USA 100:6646-6651; Hosmans et al.,2004, Hepatology 40 (Suppl. 1), 282A; and U.S. Pat. No. 6,924,271; thecontents of each are hereby incorporated by reference in theirentireties.

In another embodiment, the second agent is Celgosivir. Celgosivir is analpha-glucosidase I inhibitor and acts through host-directedglycosylation. In preclinical studies, celgosivir has demonstratedstrong synergy with IFNα plus ribavirin. See, e.g., Whitby et al., 2004,Antivir Chem Chemother. 15(3):141-51. Celgosivir is currently beingevaluated in a Phase II monotherapy study in chronic HCV patients inCanada.

In another embodiment, the second agent is ursodeoxycholic acid, or aderivative thereof (such as tauroursodeoxycholic acid, a taurineconjugate of ursodeoxycholic acid). Ursodeoxycholic acid is known to behighly effective hepatitis C virus proliferation inhibitor which givesno adverse reaction, see for example U.S. Pat. Nos. 5,846,964 and5,863,550, the contents of each are hereby incorporated by reference intheir entireties.

5.4 Pharmaceutical Compositions and Methods of Administration

The cyclosporin derivatives used in the methods of the present inventionare preferably provided using pharmaceutical compositions containing atleast one compound of general formula (I), if appropriate in the saltform, either used alone or in the form of a combination with one or morecompatible and pharmaceutically acceptable carriers, such as diluents oradjuvants, or with another anti-HCV agent.

In certain embodiments, the second agent of the invention can beformulated or packaged with the cyclosporin derivatives of theinvention. Of course, the second agent will only be formulated with thecyclosporin derivative of the present invention when, according to thejudgment of those of skill in the art, such co-formulation should notinterfere with the activity of either agent or the method ofadministration. In certain embodiments, the cyclosporin derivative ofthe invention and the second agent are formulated separately. They canbe packaged together, or packaged separately, for the convenience of thepractitioner of skill in the art.

In clinical practice the active agents of the present invention may beadministered by any conventional route, in particular orally,parenterally, rectally or by inhalation (e.g. in the form of aerosols).In certain embodiments, the cyclosporin derivatives of the presentinvention are administered orally.

Use may be made, as solid compositions for oral administration, oftablets, pills, hard gelatin capsules, powders or granules. In thesecompositions, the active product according to the invention is mixedwith one or more inert diluents or adjuvants, such as sucrose, lactoseor starch.

These compositions can comprise substances other than diluents, forexample a lubricant, such as magnesium stearate, or a coating intendedfor controlled release

Use may be made, as liquid compositions for oral administration, ofsolutions which are pharmaceutically acceptable, suspensions, emulsions,syrups and elixirs containing inert diluents, such as water or liquidparaffin. These compositions can also comprise substances other thandiluents, for example wetting, sweetening or flavoring products.

The compositions for parenteral administration can be emulsions orsterile solutions. Use may be made, as solvent or vehicle, of propyleneglycol, a polyethylene glycol, vegetable oils, in particular olive oil,or injectable organic esters, for example ethyl oleate. Thesecompositions can also contain adjuvants, in particular wetting,isotonizing, emulsifying, dispersing and stabilizing agents.Sterilization can be carried out in several ways, for example using abacteriological filter, by radiation or by heating. They can also beprepared in the form of sterile solid compositions which can bedissolved at the time of use in sterile water or any other injectablesterile medium.

The compositions for rectal administration are suppositories or rectalcapsules which contain, in addition to the active principle, excipientssuch as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

The compositions can also be aerosols. For use in the form of liquidaerosols, the compositions can be stable sterile solutions or solidcompositions dissolved at the time of use in apyrogenic sterile water,in saline or any other pharmaceutically acceptable vehicle. For use inthe form of dry aerosols intended to be directly inhaled, the activeprinciple is finely divided and combined with a water-soluble soliddiluent or vehicle, for example dextran, mannitol or lactose.

In one embodiment, a composition of the invention is a pharmaceuticalcomposition or a single unit dosage form. Pharmaceutical compositionsand single unit dosage forms of the invention comprise aprophylactically or therapeutically effective amount of one or moreprophylactic or therapeutic agents (e.g., a compound of the invention,or other prophylactic or therapeutic agent), and a typically one or morepharmaceutically acceptable carriers or excipients. In a specificembodiment and in this context, the term “pharmaceutically acceptable”means approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund'sadjuvant (complete and incomplete)), excipient, or vehicle with whichthe therapeutic is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a preferred carrier whenthe pharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E.W. Martin.

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well-known to those skilled inthe art of pharmacy, and non limiting examples of suitable excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a subjectand the specific active ingredients in the dosage form. The compositionor single unit dosage form, if desired, can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents.

Lactose free compositions of the invention can comprise excipients thatare well known in the art and are listed, for example, in the U.S.Pharmocopia (USP) SP (XXI)/NF (XVI). In general, lactose freecompositions comprise an active ingredient, a binder/filler, and alubricant in pharmaceutically compatible and pharmaceutically acceptableamounts. Exemplary lactose free dosage forms comprise an activeingredient, microcrystalline cellulose, pre gelatinized starch, andmagnesium stearate.

This invention further encompasses anhydrous pharmaceutical compositionsand dosage forms comprising active ingredients, since water canfacilitate the degradation of some compounds. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long term storage in order to determinecharacteristics such as shelf life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379 80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the inventioncan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizers,” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

The pharmaceutical compositions and single unit dosage forms can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such compositions and dosage forms willcontain a prophylactically or therapeutically effective amount of aprophylactic or therapeutic agent preferably in purified form, togetherwith a suitable amount of carrier so as to provide the form for properadministration to the subject. The formulation should suit the mode ofadministration. In a certain embodiment, the pharmaceutical compositionsor single unit dosage forms are sterile and in suitable form foradministration to a subject, for example, an animal subject, such as amammalian subject, for example, a human subject.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, intramuscular, subcutaneous,oral, buccal, sublingual, inhalation, intranasal, transdermal, topical,transmucosal, intra-tumoral, intra-synovial and rectal administration.In a specific embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous, subcutaneous, intramuscular, oral, intranasal or topicaladministration to human beings. In an embodiment, a pharmaceuticalcomposition is formulated in accordance with routine procedures forsubcutaneous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a subject,including suspensions (e.g., aqueous or non aqueous liquid suspensions,oil in water emulsions, or a water in oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a subject; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a subject.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. For example, a dosage form usedin the initial treatment of viral infection may contain larger amountsof one or more of the active ingredients it comprises than a dosage formused in the maintenance treatment of the same infection. Similarly, aparenteral dosage form may contain smaller amounts of one or more of theactive ingredients it comprises than an oral dosage form used to treatthe same disease or disorder. These and other ways in which specificdosage forms encompassed by this invention will vary from one anotherwill be readily apparent to those skilled in the art. See, e.g.,Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing, EastonPa. (2000).

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

Typical dosage forms of the invention comprise a compound of theinvention, or a pharmaceutically acceptable salt, solvate or hydratethereof lie within the range of from about 0.1 mg to about 1000 mg perday, given as a single once-a-day dose in the morning or as divideddoses throughout the day taken with food. Particular dosage forms of theinvention have about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0,15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of the activecyclosporin.

5.4.1 Oral Dosage Forms

Pharmaceutical compositions of the invention that are suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 20th ed., MackPublishing, Easton Pa. (2000).

In certain embodiments, the oral dosage forms are solid and preparedunder anhydrous conditions with anhydrous ingredients, as described indetail in the sections above. However, the scope of the inventionextends beyond anhydrous, solid oral dosage forms. As such, furtherforms are described herein.

Typical oral dosage forms of the invention are prepared by combining theactive ingredient(s) in an intimate admixture with at least oneexcipient according to conventional pharmaceutical compoundingtechniques. Excipients can take a wide variety of forms depending on theform of preparation desired for administration. For example, excipientssuitable for use in oral liquid or aerosol dosage forms include, but arenot limited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of theinvention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre gelatinized starch, hydroxypropylmethyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL PH 101, AVICEL PH 103 AVICEL RC581, AVICEL PH 105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC 581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL PH 103™ and Starch 1500LM.

Disintegrants are used in the compositions of the invention to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms of the invention. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,specifically from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, agar agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, pre gelatinized starch, otherstarches, clays, other algins, other celluloses, gums, and mixturesthereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB O SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

5.4.2 Delayed Release Dosage Forms

Active ingredients such as the compounds of the invention can beadministered by controlled release means or by delivery devices that arewell known to those of ordinary skill in the art. Examples include, butare not limited to, those described in U.S. Pat. Nos. 3,845,770;3,916,899; 3,536,809; 3,598,123; and 4,008,719; 5,674,533; 5,059,595;5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480;5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945;5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363;6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358;6,699,500 each of which is incorporated herein by reference. Such dosageforms can be used to provide slow or controlled release of one or moreactive ingredients using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, microparticles, liposomes, microspheres, or acombination thereof to provide the desired release profile in varyingproportions. Suitable controlled release formulations known to those ofordinary skill in the art, including those described herein, can bereadily selected for use with the active ingredients of the invention.The invention thus encompasses single unit dosage forms suitable fororal administration such as, but not limited to, tablets, capsules,gelcaps, and caplets that are adapted for controlled release.

All controlled release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non controlledcounterparts. Ideally, the use of an optimally designed controlledrelease preparation in medical treatment is characterized by a minimumof drug substance being employed to cure or control the condition in aminimum amount of time. Advantages of controlled release formulationsinclude extended activity of the drug, reduced dosage frequency, andincreased subject compliance. In addition, controlled releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the drug, and can thus affectthe occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

In certain embodiments, the drug may be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In one embodiment, a pump may be used(see, Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989)). In another embodiment, polymeric materials can be used. In yetanother embodiment, a controlled release system can be placed in asubject at an appropriate site determined by a practitioner of skill,i.e., thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138(1984)). Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)). The active ingredient can bedispersed in a solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl ac late copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The active ingredient then diffuses through the outerpolymeric membrane in a release rate controlling step. The percentage ofactive ingredient in such parenteral compositions is highly dependent onthe specific nature thereof, as well as the needs of the subject.

5.4.3 Parenteral Dosage Forms

Although solid, anhydrous oral dosage forms are preferred, the presentinvention also provides parenteral dosage forms. Parenteral dosage formscan be administered to subjects by various routes including, but notlimited to, subcutaneous, intravenous (including bolus injection),intramuscular, and intraarterial. Because their administration typicallybypasses subjects' natural defenses against contaminants, parenteraldosage forms are preferably sterile or capable of being sterilized priorto administration to a subject. Examples of parenteral dosage formsinclude, but are not limited to, solutions ready for injection, dryproducts ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection, andemulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms of the invention.

5.4.4 Transdermal, Topical & Mucosal Dosage Forms

Although solid, anhydrous oral dosage forms are preferred, the presentinvention also provides transdermal, topical, and mucosal dosage forms.Transdermal, topical, and mucosal dosage forms of the invention include,but are not limited to, ophthalmic solutions, sprays, aerosols, creams,lotions, ointments, gels, solutions, emulsions, suspensions, or otherforms known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th), 18th and 20^(th) eds., MackPublishing, Easton Pa. (1980, 1990 & 2000); and Introduction toPharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia(1985). Dosage forms suitable for treating mucosal tissues within theoral cavity can be formulated as mouthwashes or as oral gels. Further,transdermal dosage forms include “reservoir type” or “matrix type”patches, which can be applied to the skin and worn for a specific periodof time to permit the penetration of a desired amount of activeingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms encompassed by this invention are well known to those skilled inthe pharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3diol, isopropyl myristate, isopropyl palmitate, mineral oil, andmixtures thereof to form lotions, tinctures, creams, emulsions, gels orointments, which are non toxic and pharmaceutically acceptable.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th), 18th and 20^(th) eds., MackPublishing, Easton Pa. (1980, 1990 & 2000).

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients of the invention. For example, penetrationenhancers can be used to assist in delivering the active ingredients tothe tissue. Suitable penetration enhancers include, but are not limitedto: acetone; various alcohols such as ethanol, oleyl, andtetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery enhancing orpenetration enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

5.4.5 Dosage and Unit Dosage Forms

In human therapeutics, the doctor will determine the posology which heconsiders most appropriate according to a preventive or curativetreatment and according to the age, weight, stage of the infection andother factors specific to the subject to be treated. Generally, dosesare from about 1 to about 1000 mg per day for an adult, or from about 5to about 250 mg per day or from about 10 to 50 mg per day for an adult.In certain embodiments, doses are from about 5 to about 400 mg per day,and more preferably 25 to 200 mg per day per adult. Dose rates of fromabout 50 to about 500 mg per day are also preferred.

In further aspects, the present invention provides methods of treatingor preventing hepatitis C virus infection in a subject by administering,to a subject in need thereof, an effective amount of a compound of theinvention, or a pharmaceutically acceptable salt thereof, with a hightherapeutic index against hepatitis C virus. The therapeutic index canbe measured according to any method known to those of skill in the art,such as the method described in the examples below. In certainembodiments, the therapeutic index is the ratio of a concentration atwhich the compound is toxic, to the concentration that is effectiveagainst hepatitis C virus. Toxicity can be measured by any techniqueknown to those of skill including cytotoxicity (e.g. IC₅₀ or IC₉₀) andlethal dose (e.g. LD₅₀ or LD₉₀). Likewise, effective concentrations canbe measured by any technique known to those of skill including effectiveconcentration (e.g. EC₅₀ or EC₉₀) and effective dose (e.g. ED₅₀ orED₉₀). Preferably, similar measurements are compared in the ratio (e.g.IC₅₀/EC₅₀, IC₉₀/EC₉₀, LD₅₀/ED₅₀ or LD₉₀/ED₉₀). In certain embodiments,the therapeutic index can be as high as 2.0, 5.0, 10.0, 15.0, 20.0,25.0, 50.0, 75.0, 100.0, 125.0, 150.0 or higher.

The amount of the compound or composition of the invention which will beeffective in the prevention or treatment of a disorder or one or moresymptoms thereof will vary with the nature and severity of the diseaseor condition, and the route by which the active ingredient isadministered. The frequency and dosage will also vary according tofactors specific for each subject depending on the specific therapy(e.g., therapeutic or prophylactic agents) administered, the severity ofthe disorder, disease, or condition, the route of administration, aswell as age, body, weight, response, and the past medical history of thesubject. Effective doses may be extrapolated from dose-response curvesderived from in vitro or animal model test systems.

Exemplary doses of a composition include milligram or microgram amountsof the active compound per kilogram of subject or sample weight (e.g.,about 10 micrograms per kilogram to about 50 milligrams per kilogram,about 100 micrograms per kilogram to about 25 milligrams per kilogram,or about 100 microgram per kilogram to about 10 milligrams perkilogram). For compositions of the invention, the dosage administered toa subject is typically 0.140 mg/kg to 3 mg/kg of the subject's bodyweight, based on weight of the active compound. In certain embodiments,the dosage administered to a subject is between 0.20 mg/kg and 2.00mg/kg, or between 0.30 mg/kg and 1.50 mg/kg of the subject's bodyweight.

In general, the recommended daily dose range of a composition of theinvention for the conditions described herein lie within the range offrom about 0.1 mg to about 1000 mg per day, given as a single once-a-daydose or as divided doses throughout a day. In one embodiment, the dailydose is administered twice daily in equally divided doses. Specifically,a daily dose range should be from about 10 mg to about 200 mg per day,more specifically, between about 10 mg and about 150 mg per day, or evenmore specifically between about 25 and about 100 mg per day. It may benecessary to use dosages of the active ingredient outside the rangesdisclosed herein in some cases, as will be apparent to those of ordinaryskill in the art. Furthermore, it is noted that the clinician ortreating physician will know how and when to interrupt, adjust, orterminate therapy in conjunction with subject response.

Different therapeutically effective amounts may be applicable fordifferent diseases and conditions, as will be readily known by those ofordinary skill in the art. Similarly, amounts sufficient to prevent,manage, treat or ameliorate such disorders, but insufficient to cause,or sufficient to reduce, adverse effects associated with the compositionof the invention are also encompassed by the above described dosageamounts and dose frequency schedules. Further, when a subject isadministered multiple dosages of a composition of the invention, not allof the dosages need be the same. For example, the dosage administered tothe subject may be increased to improve the prophylactic or therapeuticeffect of the composition or it may be decreased to reduce one or moreside effects that a particular subject is experiencing.

In a specific embodiment, the dosage of the composition of the inventionor a composition of the invention, based on weight of the activecompound, administered to prevent, treat, manage, or ameliorate adisorder, or one or more symptoms thereof in a subject is 0.1 mg/kg, 1mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10 mg/kg, or 15mg/kg or more of a subject's body weight. In another embodiment, thedosage of the composition of the invention or a composition of theinvention administered to prevent, treat, manage, or ameliorate adisorder, or one or more symptoms thereof in a subject is a unit dose of0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg,0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg,1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

In certain embodiments, treatment or prevention can be initiated withone or more loading doses of a compound or composition of the inventionfollowed by one or more maintenance doses. In such embodiments, theloading dose can be, for instance, about 60 to about 400 mg per day, orabout 100 to about 200 mg per day for one day to five weeks. The loadingdose can be followed by one or more maintenance doses. Each maintenancedoes can be, independently, about from about 10 mg to about 200 mg perday, more specifically, between about 25 mg and about 150 mg per day, oreven more specifically between about 25 and about 80 mg per day.Maintenance doses are preferably administered daily and can beadministered as single doses, or as divided doses.

In certain embodiments, a dose of a compound or composition of theinvention can be administered to achieve a steady-state concentration ofthe active ingredient in blood or serum of the subject. The steady-stateconcentration can be determined by measurement according to techniquesavailable to those of skill or can be based on the physicalcharacteristics of the subject such as height, weight and age. Incertain embodiments, a sufficient amount of a compound or composition ofthe invention is administered to achieve a steady-state concentration inblood or serum of the subject of from about 300 to about 4000 ng/mL,from about 400 to about 1600 ng/mL, or from about 600 to about 1200ng/mL. Loading doses can be administered to achieve steady-state bloodor serum concentrations of about 1200 to about 8000 ng/mL, or about 2000to about 4000 ng/mL for one to five days. Maintenance doses can beadministered to achieve a steady-state concentration in blood or serumof the subject of from about 300 to about 4000 ng/mL, from about 400 toabout 1600 ng/mL, or from about 600 to about 1200 ng/mL.

In certain embodiments, administration of the same composition of theinvention may be repeated and the administrations may be separated by atleast 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days,2 months, 75 days, 3 months, or 6 months. In other embodiments,administration of the same prophylactic or therapeutic agent may berepeated and the administration may be separated by at least at least 1day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2months, 75 days, 3 months, or 6 months.

In certain aspects, the present invention provides unit dosagescomprising a compound of the invention, or a pharmaceutically acceptablesalt thereof, in a form suitable for administration. Such forms aredescribed in detail above. In certain embodiments, the unit dosagecomprises 1 to 1000 mg, 5 to 250 mg or 10 to 50 mg active ingredient. Inparticular embodiments, the unit dosages comprise about 1, 5, 10, 25,50, 100, 125, 250, 500 or 1000 mg active ingredient. Such unit dosagescan be prepared according to techniques familiar to those of skill inthe art.

The dosages of the second agents are to be used in the combinationtherapies of the invention. In certain embodiments, dosages lower thanthose which have been or are currently being used to prevent or treatHCV infection are used in the combination therapies of the invention.The recommended dosages of second agents can obtained from the knowledgeof those of skill. For those second agents that are approved forclinical use, recommended dosages are described in, for example, Hardmanet al., eds., 1996, Goodman & Gilman's The Pharmacological Basis OfBasis Of Therapeutics 9^(th) Ed, Mc-Graw-Hill, N.Y.; Physician's DeskReference (PDR) 57^(th) Ed., 2003, Medical Economics Co., Inc.,Montvale, N.J., which are incorporated herein by reference in itsentirety.

In various embodiments, the therapies (e.g., the cyclosporin derivativeof the invention and the second agent) are administered less than 5minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hourapart, at about 1 to about 2 hours apart, at about 2 hours to about 3hours apart, at about 3 hours to about 4 hours apart, at about 4 hoursto about 5 hours apart, at about 5 hours to about 6 hours apart, atabout 6 hours to about 7 hours apart, at about 7 hours to about 8 hoursapart, at about 8 hours to about 9 hours apart, at about 9 hours toabout 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours part. In certainembodiments, two or more therapies are administered within the samepatent visit.

In certain embodiments, the cyclosporin derivative of the invention andthe second agent are cyclically administered. Cycling therapy involvesthe administration of a first therapy (e.g., a first prophylactic ortherapeutic agents) for a period of time, followed by the administrationof a second therapy (e.g., a second prophylactic or therapeutic agents)for a period of time, followed by the administration of a third therapy(e.g., a third prophylactic or therapeutic agents) for a period of timeand so forth, and repeating this sequential administration, i.e., thecycle in order to reduce the development of resistance to one of theagents, to avoid or reduce the side effects of one of the agents, and/orto improve the efficacy of the treatment.

In certain embodiments, administration of the same agent may be repeatedand the administrations may be separated by at least 1 day, 2 days, 3days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months. In other embodiments, administration of the sameagent may be repeated and the administration may be separated by atleast at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days,45 days, 2 months, 75 days, 3 months, or 6 months.

In certain embodiments, a cyclosporin derivative of the invention and asecond agent are administered to a patient, preferably a mammal, morepreferably a human, in a sequence and within a time interval such thatthe cyclosporin derivative can act together with the other agent toprovide an increased benefit than if they were administered otherwise.For example, the second active agent can be administered at the sametime or sequentially in any order at different points in time; however,if not administered at the same time, they should be administeredsufficiently close in time so as to provide the desired therapeutic orprophylactic effect. In one embodiment, the cyclosporin derivative andthe second active agent exert their effect at times which overlap. Eachsecond active agent can be administered separately, in any appropriateform and by any suitable route. In other embodiments, the cyclosporinderivative is administered before, concurrently or after administrationof the second active agent.

In various embodiments, the cyclosporin derivative and the second agentare administered less than about 1 hour apart, at about 1 hour apart, atabout 1 hour to about 2 hours apart, at about 2 hours to about 3 hoursapart, at about 3 hours to about 4 hours apart, at about 4 hours toabout 5 hours apart, at about 5 hours to about 6 hours apart, at about 6hours to about 7 hours apart, at about 7 hours to about 8 hours apart,at about 8 hours to about 9 hours apart, at about 9 hours to about 10hours apart, at about 10 hours to about 11 hours apart, at about 11hours to about 12 hours apart, no more than 24 hours apart or no morethan 48 hours apart. In other embodiments, the cyclosporin derivativeand the second agent are administered concurrently.

In other embodiments, the cyclosporin derivative and the second agentare administered at about 2 to 4 days apart, at about 4 to 6 days apart,at about 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart.

In certain embodiments, the cyclosporin derivative and the second agentare cyclically administered to a patient. Cycling therapy involves theadministration of a first agent for a period of time, followed by theadministration of a second agent and/or third agent for a period of timeand repeating this sequential administration. Cycling therapy can reducethe development of resistance to one or more of the therapies, avoid orreduce the side effects of one of the therapies, and/or improve theefficacy of the treatment.

In certain embodiments, the cyclosporin derivative and the second activeagent are administered in a cycle of less than about 3 weeks, about onceevery two weeks, about once every 10 days or about once every week. Onecycle can comprise the administration of a cyclosporin derivative andthe second agent by infusion over about 90 minutes every cycle, about 1hour every cycle, about 45 minutes every cycle. Each cycle can compriseat least 1 week of rest, at least 2 weeks of rest, at least 3 weeks ofrest. The number of cycles administered is from about 1 to about 12cycles, more typically from about 2 to about 10 cycles, and moretypically from about 2 to about 8 cycles.

In other embodiments, courses of treatment are administered concurrentlyto a patient, i.e., individual doses of the second agent areadministered separately yet within a time interval such that thecyclosporin derivative can work together with the second active agent.For example, one component can be administered once per week incombination with the other components that can be administered onceevery two weeks or once every three weeks. In other words, the dosingregimens are carried out concurrently even if the therapeutics are notadministered simultaneously or during the same day.

The second agent can act additively or synergistically with thecyclosporin derivative. In one embodiment, a cyclosporin derivative isadministered concurrently with one or more second agents in the samepharmaceutical composition. In another embodiment, a cyclosporinderivative is administered concurrently with one or more second agentsin separate pharmaceutical compositions. In still another embodiment, acyclosporin derivative is administered prior to or subsequent toadministration of a second agent. The invention contemplatesadministration of a cyclosporin derivative and a second agent by thesame or different routes of administration, e.g., oral and parenteral.In certain embodiments, when a cyclosporin derivative is administeredconcurrently with a second agent that potentially produces adverse sideeffects including, but not limited to, toxicity, the second active agentcan advantageously be administered at a dose that falls below thethreshold that the adverse side effect is elicited.

5.5 Kits

The invention also provides kits for use in methods of treatment orprophylaxis of HCV infection. The kits can include a cyclosporinderivative compound or composition of the invention, a second agent orcomposition, and instructions providing information to a health careprovider regarding usage for treating or preventing a bacterialinfection. Instructions may be provided in printed form or in the formof an electronic medium such as a floppy disc, CD, or DVD, or in theform of a website address where such instructions may be obtained. Aunit dose of a cyclosporin derivative or composition of the invention,or a second agent or composition, can include a dosage such that whenadministered to a subject, a therapeutically or prophylacticallyeffective plasma level of the compound or composition can be maintainedin the subject for at least 1 days. In some embodiments, a compound orcomposition can be included as a sterile aqueous pharmaceuticalcomposition or dry powder (e.g., lyophilized) composition. In oneembodiment, the compound is according to formula (I).

In some embodiments, suitable packaging is provided. As used herein,“packaging” refers to a solid matrix or material customarily used in asystem and capable of holding within fixed limits a cyclosporinderivative of the invention and/or a second agent suitable foradministration to a subject. Such materials include glass and plastic(e.g., polyethylene, polypropylene, and polycarbonate) bottles, vials,paper, plastic, and plastic-foil laminated envelopes and the like. Ife-beam sterilization techniques are employed, the packaging should havesufficiently low density to permit sterilization of the contents.

The following Examples illustrate the synthesis of representativecyclosporin derivatives used in the present invention. These examplesare not intended, nor are they to be construed, as limiting the scope ofthe invention. It will be clear that the invention may be practicedotherwise than as particularly described herein. Numerous modificationsand variations of the present invention are possible in view of theteachings herein and, therefore, are within the scope of the invention.

6. EXAMPLES 6.1 Example 13-Methoxy-4-(gamma-hydroxymethylleucine)-cyclosporin3-Methoxy-4-(gamma-hydroxymethylleucine)-cyclosporin

To a solution of1,4-di-acetyl-3-methoxy-4-(gamma-hydroxymethylleucine)-cyclosporin (275mg) in methanol (15 mL) was added 25 wt % sodium methoxide in methanol(0.12 mL) and the resulting mixture was stirred at room temperature for24 h under nitrogen. Methanol was removed under reduced pressure and theresidue was diluted with ethyl acetate (50 mL), washed with saturatedammonium chloride (30 mL), brine (30 mL), and dried over anhydroussodium sulfate. After solvent removal, the residue was purified usingpreparative liquid chromatography to yield 33 mg the title compound(Compound T).

NMR signals for this compound in deuterochloroform are at (ppm) 5.80(singlet, sarcosine H), and four doublet NH signals at 7.14, 7.39, 7.69,and 7.96. LCMS (ESI): calcd for C₆₃H₁₁₃N₁₁O₁₄: 1247, found 1248.5(M+H)⁺.

3-[(R)-2-(N,N-dimethylamino)ethylthio-Sar]-4-(gammahydroxymethyl-leucine)cyclosporin

3-[(R)-2-(N,N-dimethylamino)ethylthio-Sar]-4-(gamma-hydroxymethyl-leucine)cyclosporin(Compound O) is prepared as described, for example, in U.S. Pat. No.5,977,067.

6.2 Example 2 HCV Activity

The instant example demonstrates that compounds of the invention areeffective against HCV infection. In addition, the instant exampledemonstrates that compounds of the invention show advantageous efficacy,or cytotoxicity, or both when compared to cyclosporin A.

The compounds of the present invention were tested for activity againstHCV using the methods adapted from those described by Kriger et al.,2001, Journal of Virology, 75:4614-4624, Pietschmann et al., 2002,Journal of Virology 76:4008-4021, and using HCV RNA constructs asdescribed in U.S. Pat. No. 6,630,343. The contents of these referencesare hereby incorporated by reference in their entireties.

Compounds were examined in the human hepatoma cell line ET (lub ubineo/ET), a HCV RNA replicon containing a stable luciferase (LUC)reporter. The HCV RNA replicon ET contains the 5′ end of HCV (with theHCV Internal Ribosome Entry Site (IRES) and the first few amino acids ofthe HCV core protein) which drives the production of a fireflyluciferase (LUC), ubiquitin, and neomycin phosphotransferase (NeoR)fusion protein. Ubiquitin cleavage releases the LUC and NeoR proteins.The EMCV IRES element controls the translation of the HCV, structuralproteins NS3-NS5. The NS3 protein cleaves the HCV polyprotein to releasethe mature NS3, NS4A, NS4B, NS5A and NS5B proteins that are required forHCV replication. At the 3′ end of the replicon is the authentic 3′ NTRof HCV. The activity of the LUC reporter is directly proportional to HCVreplication levels and positive-control antiviral compounds produce areproducible antiviral response using the LUC endpoint.

The compounds were dissolved in DMSO at five half-log concentrationseach, ranging from either 0.02 to 2.0 μM, 0.03 to 3 μM, 2.0 to 20 μM, or1 to 100 μM. Subconfluent cultures of the ET line were plated out into96 well plates dedicated for the analysis of cell numbers (cytotoxicity)or antiviral activity and the next day the compounds were added to theappropriate wells. The cells were processed 72 h later when the cellswere still subconfluent. Antiviral activity was expressed as EC₅₀ andEC₉₀, the effective concentration of compound that reduced viralreplication by 50% and 90%, respectively. Compound EC₅₀ and EC₉₀ valueswere derived from HCV RNA levels assessed as HCV RNA replicon derivedLUC activity. Cytotoxicity was expressed as IC₅₀ and IC₉₀, theconcentration of compound that inhibited cell viability by 50% and 90%,respectively. Compound IC₅₀ and IC₉₀ values were calculated using acolorimetric assay as an indication of cell numbers and cytotoxicity.The activity of the LUC reporter is directly proportional to HCV RNAlevels in the human cell line. The HCV-replicon assay was validated inparallel experiments using interferon-alpha-2b as a positive control.Cyclosporine was also tested by way of comparison. Compounds of theinvention potently inhibit HCV replication in human liver cells to agreater extent than cyclosporin. In addition, when considering the levelof cytotoxicity, many of the compounds of this invention exhibit a widersafety margin (for example, cytotoxicity IC₅₀ versus antiviral EC₅₀)than cyclosporine.

The results were as follows (unless otherwise stated all values areexpressed in nM), “N/D” means that the data was not determined. HCVActivity Cytotoxicity Compound EC50 EC90 IC50 IC90 O 122 30013440 >20000 T 80 410 >2000 >2000 Cyclosporine 400 1420 5780 19403

6.3 Example 3 Cyclophilin Binding and HCV Activity

The instant example provides further methods for evaluating theeffectiveness of compounds of the invention for treating or preventingHCV infection in a subject in need thereof.

It has been demonstrated that certain cyclosporins are effective intreating or preventing HCV infection through the binding of thecyclosporin to cyclophilin B (CyPB). See Watashi et al., 2005, MolecularCell 19:111-122; Nakagawa et al., 2005 Gastroenterology 129(3):1031-41;the contents of which are hereby incorporated by reference in theirentirety. Although not intending to be bound by any particular theory ofoperation, it is believed that cyclophilin B is critical for theefficient replication of the HCV genome. Cyclosporin A and derivativesof cyclosporin that inhibit cyclophilin B can dramatically reduce thereplication of HCV in standard assays.

Accordingly, compounds of the present invention are shown to beeffective for the treatment or prevention of HCV infection by evaluatingtheir binding or modulation of cyclophilin, for instance cyclophilin B.Modulation of cyclophilin by a compound of this invention is measuredaccording to standard techniques, for example, those described inWatashi et al., 2005, those described in Nakagawa et al., 2005, or thosedescribed in Billich et al., J. Virol. 69:2451-2461, the contents ofwhich are hereby incorporated by reference in their entireties.

6.4 Example 4 Oral Dosage Forms

One or more of the compounds of the invention can be formulated as acapsule. Such a capsule can comprise 10 to 200 mg of the compound and onor more excipients selected from the group consisting ofmicrocrystalline cellulose, pregelatinized starch, lactose, sodiumstarch glycolate, crospovidone, povidone, hydroxypropylcellulose,magnesium stearate and silicon dioxide. The resulting composition can beencapsulated with one or more standard encapsulation compositions suchas gelatin or a plasticizer.

One or more of the compounds of the invention can be formulated as asalt in a syrup or elixir. The compound or compounds can be at a totalconcentration of 5 to 50 mg/mL. The syrup or elixir can further comprisepolyethylene glycol, propylene glycol, mixtures of polyethylene glycol,PEG 400, a block copolymer of ethylene oxide and propylene oxide (e.g.,poloxamer 407), polysorbate 20, ethanol, a sugar, citric acid and/orflavoring.

6.5 Example 5 Additive and Synergistic anti-HCV Activity of Compounds ofthe Invention in Combination with Interferon

Compounds and combinations were evaluated in the HCV replicon assaydescribed in Example 2 with changes indicated below.

For single-compound assays, the compounds were dissolved in DMSO at fivehalf-log concentrations each, ranging from either 0.02 to 2 μM or 0.2 to20 μM. Compound EC₅₀ and EC₉₀ values, as well as IC₅₀ and IC₉₀ values,were determined according to Example 2. The HCV-replicon assay wasvalidated in parallel experiments using interferon-alpha-2b as apositive control. Cyclosporine A was also tested.

The HCV RNA replicon antiviral evaluation assay was utilized to examinethe efficacy and cytotoxicity of two compounds (e.g., Drug 1 and Drug 2)in combinations of five versus nine half-log concentrations. The assaywas performed using a microtiter plate format for allocation of media,drug, cells, and virus. Sub-confluent cultures of the ET line weretransferred into 96-well plates for the analysis of cell numbers(cytotoxicity) or antiviral activity; approximately 24 h later Drugs 1and 2 were added to the appropriate wells. Cells were processed 72 hrlater when the cells were still sub-confluent. The HCV RNA repliconlevels were assessed as HCV RNA replicon-derived Luc activity asdescribed in Example 2. The cytotoxicity was evaluated as theconcentration of the drug that reduced cell numbers as described inExample 2.

Compounds of this invention were tested as “Drug 2” at nineconcentrations between 0.0078 to 2.0 μM. Interferon-alpha (Sigma) wastested as Drug 1 at 0.005 to 0.5 international units per mL (IU/mL). Thedata obtained from these checkerboard assays were analyzed with theMacSynergy II (v2.01) (Prichard & Shipman, 1990, Antiviral Res.14:181-205) and Delta Graph (v1.5d) programs. Each data point used tocreate three-dimensional plots (FIGS. 1A-1C) was derived from the resultof triplicate samples. The statistical relevance of the data wasanalyzed and plotted at one of three confidence levels (68%, 95% or 99%)to display combinatorial results as additive, synergistic orantagonistic.

Effects of the drug combinations were calculated based on the activityof each compound when tested alone. The expected additive antiviralprotection was subtracted from the experimentally determined antiviralactivity at each combination concentration resulting in a positive value(synergy), a negative value (antagonism) or zero (additive). The resultspresented three dimensionally at each combination concentration yield asurface of activity extending above (synergy) or below (antagonism) the“plane of additivity.” Synergy is defined as drug combinations yieldingsynergy volumes greater than 50. Slightly synergistic activity andhighly synergistic activity have been operationally defined as yieldingsynergy volumes of 50-100 and >100, respectively. Additive druginteractions have synergy volumes in the range of −50 to 50.Antagonistic drug interactions have synergy volumes in the range or —50and —100, and those <−100 are highly antagonistic.

For Compound O, Compound T, and cyclosporin A, the volume of the surfacewas calculated and expressed as a synergy volume (in units ofconcentration times concentration times percent; e.g. μM²%, nM²%, nMμM%, and for IFN (IU/ml)μM %, etc.) at the 95% confidence interval (FIGS.1A-1C).

As shown in FIG. 1A, the combination of Compound O and IFNα is highlysynergistic. The EC₅₀ of Compound O alone was 100 nM, and the IC₅₀ ofCompound O alone was >2000 nM. At a 95% confidence interval, theantiviral synergy volume was 122 IU/mLμM %, or highly synergistic.

As shown in FIG. 1B, the combination of Compound T and IFNα is additive.The EC₅₀ of Compound T alone was 200 nM, and the IC₅₀ of Compound Talone was >2000 nM. At a 95% confidence interval, the antiviral synergyvolume was 6 IU/mLμM %, or additive.

As shown in FIG. 1C, the combination of Cyclosporin A and IFNα isadditive. The EC₅₀ of Cyclosporin A alone was 750 nM, and the IC₅₀ ofCyclosporin A alone was >2000 nM. At a 95% confidence interval, theantiviral synergy volume was 43 IU/mLμM %, or additive.

6.6 Example 6 Activity against HCV of Compounds of the Invention inCombination with a Reverse Transcriptase Inhibitor

Compounds and combinations were evaluated in the HCV replicon assaydescribed in Example 5 with changes noted below.

Compound O of this invention were tested as “Drug 2” at nineconcentrations between 0.0078 to 2.0 μM. 2′-C-methyl-cytidine (theactive form of the prodrug valopicitabine, and a potent NS5B inhibitor)was added as Drug 1; Drug 1 was tested at 62.5, 125, 250, and 500 nM.

For Compound O, the volume of the surface was calculated and expressedas a synergy volume (in units of concentration times concentration timespercent; e.g. μM²%.) at the 95% confidence interval (FIG. 2).

As shown in FIG. 2, the combination of Compound O and2′-C-methyl-cytidine is highly synergistic. In this experiment, the EC₅₀values of Compound O and 2′-C-methyl-cytidine alone were 250 nM and 750nM, respectively. The IC₅₀ of Compound O alone was >2000 nM. At a 95%confidence interval, the antiviral synergy volume was 228 μM²%, orhighly synergistic.

6.7 Example 7 Activity Against HCV and Toxicity of Compounds of theInvention in Combination with a Protease Inhibitor

Compounds and combinations were evaluated in the HCV replicon assaydescribed in Example 5 with changes noted below. (1R, 2S,5S)-3-Azabicyclo[3,1,0]hexane-2-carboxamide,N-[3-amino-1-cyclobutylmethyl)-2,3-dioxopropyl]-3-[(2S)-2-[[[1,1-dimethylethyl]amino]carbonylamino]-3,3-dimethyl-1-oxobutyl]-6,6-dimethyl,a protease inhibitor (a HCV NS3-4A protease inhibitor), hereinafterreferred to as Compound 3, was prepared according the methods describedin US patent application publication No. 2005/0249702 and PCTpublication No. WO2004/113294, the contents of which are incorporated byreference herein and relied upon (the compound was tested as a 1:1diastereomeric mixture at the chiral center bonded to cyclobutylmethylmoiety: i.e., carbon-atom 1 of the3-amino-1-cyclobutylmethyl)-2,3-dioxopropyl residue.)

Effects of the drug combinations were calculated based on the activityof each compound when tested alone. The expected additive antiviralprotection was subtracted from the experimentally determined antiviralactivity at each combination concentration resulting in a positive value(synergy), a negative value (antagonism), or zero (additivity). Theresults of the combination assays are presented three dimensionally ateach combination concentration, yielding a surface of activity extendingabove (synergy) or below (antagonism) the plane of additivity. Thevolume of the surface is calculated and expressed as a synergy volume(in units of concentration times concentration times percent; e.g. μM2%,nM2%, nMμM %, etc.) calculated at the 95% confidence interval. For thesestudies, synergy is defined as drug combinations yielding synergyvolumes greater than 50. Additive drug interactions have synergy volumesin the range of −50 to 50, while synergy volumes between −50 and —100are considered slightly antagonistic and those <−100 are highlyantagonistic.

As shown in FIG. 3A the combination of Compound O and Compound 3 wasadditive or mildly synergistic.

As shown in FIG. 3B, the combination of Compound O and Compound 3 showedantagonistic cytotoxicity, i.e. a reduced toxicity.

6.8 Example 8 Activity of Cyclosporin Derivatives Against Bovine ViralDiarrhea Virus in Combination Studies with Interferon and Ribavirin

Compounds were evaluated alone and in combination with ribavirin oralpha interferon (IFN-α) in Madin-Darby bovine kidney cells against theNADL (National Animal Disease Laboratory) strain of the HCV surrogate,Bovine Viral Diarrhea Virus (BVDV). BVDV, also a member of theFlaviviridae family of viruses, shares many important features with HCVand is recognized as a valid surrogate for the evaluation of antiviralcompounds for use against HCV (Buckwald V E, Wei J, Wenzel-Mathers M,Russell J. “Synergistic in vitro interactions between alpha interferonand ribavirin against bovine viral diarrhea virus and yellow fever virusas surrogate models of hepatitis C virus replication” Antimicrob AgentsChemother. 2003, 47: 2293). BVDV and HCV have similar genomic RNAstructures, and both utilize an internal ribosome entry site (IRES) fortranslation initiation. Using the well established host-cell line knownas Madin-Darby bovine kidney (MDBK) cells, compounds were tested againstBVDV, alone and in combination with interferon and ribavirin.

The methodology used herein for testing compounds as single agents or indrug combinations against BVDV's cytopathic effect in MDBK cells hasbeen previously described: Buckwald V E, et al. Antimicrob AgentsChemother. 2003, 47: 2293). A summary of the methods is provided. Thecompounds of this invention were dissolved in DMSO to prepared stocksolutions for dilution into the assay. Compounds were serially diluted,starting at a high concentration of 10 or 20 μM, in half-logarithmicincrements for evaluation in the in vitro antiviral assay. As singleagents, Ribavirin (Sigma, St. Louis, Mo.) and alpha interferon-alpha(IFN-α, Sigma) were evaluated at 100 μM and 10 U/ml high testconcentration, respectively, and serially diluted in half-logarithmicincrements for evaluation in the in vitro antiviral assay. MDBK cellswere suspended at 5×10⁴ cells per ml in tissue culture medium and addedto flat bottom microtiter plates in a volume of 100 μL. Total cells andcell viability quantification was performed using a hemacytometer andTrypan Blue dye exclusion. The plates were incubated at 37° C./5% CO₂overnight to allow for cell adherence.

BVDV was obtained from the American Type Culture Collection (ATCC) andwas grown in MDBK cells for the production of stock virus pools. Viruswas suspended and diluted into tissue culture medium such that theamount of virus added to each well in a volume of 100 μL was the amountdetermined to yield 85 to 95% cell killing at 6 days post-infection.

Each microtiter plate contained cell control wells (cells only), viruscontrol wells (cells plus virus), drug toxicity wells (cells pluscompound only), drug colorimetric control wells (drug only), and theexperimental wells (compound plus cells plus virus). Samples were testedin triplicate for efficacy and in duplicate for cytotoxicity. Followingincubation at 37° C. in a 5% CO₂ incubator, the test plates were stainedwith the tetrazolium dye XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymesof metabolically active cells to a soluble formazan product, allowingrapid quantitative analysis of the inhibition of BVDV induced cellkilling by anti-BVDV test substances. XTT solution was prepared daily asa stock of 1 mg/mL in medium (RPMI1640). Phenazine methosulfate (PMS)solution was prepared at 0.15 mg/mL in PBS and stored in the dark at−20° C. XTT/PMS stock was prepared immediately before use by adding 40μL of PMS per ml of XTT solution. Fifty microliters of XTT/PMS was addedto each well of the plate and the plate was reincubated for 4 hours at37° C. Plates were sealed with adhesive plate sealers and shaken gentlyor inverted several times to mix the soluble formazan product and theplate was read spectrophotometrically at 450/650 nm with a MolecularDevices Vmax plate reader.

Data Analysis—Raw data was collected from the Softmax Pro 4.6 softwareand imported into a Microsoft Excel 2003 spreadsheet for analysis bylinear curve fit calculations. Antiviral activity was expressed as EC₅₀and EC₉₀, the effective concentration of compound that reduced viralreplication by 50% and 90%, respectively; cytotoxicity was expressed asIC₅₀ and IC₉₀, the concentration of compound that inhibited cellviability by 50% and 90%, respectively. Data was summarized in tables ofmean optical density values at each compound concentration in theefficacy, toxicity and colorimetric wells along with calculatedpercentage of reduction values for viral cytopathic effect (CPE) andcell viability; data was used for graphical presentation of thepercentage of cell viability and percentage of reduced viral CPE at eachtest concentration.

Drug-Drug Combination Experiments

The BVDV antiviral evaluation assay was utilized to examine the efficacyand cytotoxicity of two compounds (e.g., Drug 1 and Drug 2) incombinations of five versus nine half-log concentrations. The assay wasperformed using a microtiter plate format for allocation of media, drug,cells, and virus. Statistical evaluations were performed according tothe method of Prichard and Shipman (Antiviral Research 1990,14:181-206). Five concentrations of each test compound (Drug 1) weretested in all possible combinations with nine concentrations of eitherribavirin or IFN-α (Drug 2). Endpoint quantification was performed byXTT-tetrazolium staining to quantify cell viability. Effects of the drugcombination were calculated based on the activity of the two compoundswhen tested alone. The expected additive antiviral protection wassubtracted from the experimentally determined antiviral activity at eachcombination concentration resulting in a positive value (synergy), anegative value (antagonism), or zero (additivity). The results of thecombination assays were expressed as a synergy volume (μM²%) calculatedat the 95% confidence interval.

For the BVDV studies, synergy was defined as drug combinations yieldingsynergy volumes greater than 50 μM²%. Slightly synergistic activity andhighly synergistic activity have been defined as yielding synergyvolumes of 50-100 μM²% and >100 μM²%, respectively. Synergy volumesbetween −50 and 50 μM²% were considered additive and synergy volumesless than −50 μM²% were considered antagonistic.

Results from drug combinations in BVDV Assay with Ribavirin:

Compounds of the invention and ribavirin were synergistic with respectto antiviral activity against BVDV, the surrogate virus for human HCV.There was no significant antagonistic activity observed when compoundsof the invention were tested in combination with ribavirin. Compounds Cand O were more synergistic than cyclosporin A, having synergy volumesof 25% greater than that of Cyclosporin A.

Results from drug combinations in BVDV Assay with IFN-α.

Additive interactions of anti-BVDV were demonstrated with compound C orCsA in combination with IFN-α. Compound O exhibited a synergisticinteraction with IFN-α. Antagonistic cytotoxicity resulted from thecombination of compound O plus IFN-α, or compound C plus IFN-α, thusindicating a reduction in toxicity to the target cells with thesecompounds when used in combination compared to the toxicity of eachcompound alone. Synergistic toxicity, i.e. an increase in toxicity incombination compared to each compound alone at those concentrations, wasobserved with CsA plus IFN-α.

All publications and patent, applications cited in this specificationare herein incorporated by reference as if each individual publicationor patent application were specifically and individually indicated to beincorporated by reference. While the invention has been described interms of various preferred embodiments, the skilled artisan willappreciate that various modifications, substitutions, omissions, andchanges may be made without departing from the spirit thereof.Accordingly, it is intended that the scope of the present invention belimited solely by the scope of the following claims, includingequivalents thereof.

1. A method for treating or preventing hepatitis C virus infection in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a cyclosporin derivativeof general formula (I):

wherein: A is residue of formula (IIa) or (IIb):

B is ethyl, 1-hydroxyethyl, isopropyl, or n-propyl; R¹ is: straight- orbranched-chain alkyl containing from one to six carbon atoms, optionallysubstituted by one or more groups R³ which may be the same or different;straight- or branched-chain alkenyl containing from two to six carbonatoms optionally substituted by one or more groups which may be the sameor different selected from the group consisting of halogen, hydroxy,amino, monoalkylamino and dialkylamino; straight- or branched-chainalkynyl containing from two to six carbon atoms, optionally substitutedby one or one or more groups which may be the same or different selectedfrom the group consisting of halogen, hydroxy, amino, monoalkylamino anddialkylamino; cycloalkyl containing from three to six carbon atomsoptionally substituted by one or more groups which may be the same ordifferent selected from the group consisting of halogen, hydroxy, amino,monoalkylamino and dialkylamino; straight- or branched-chainalkoxycarbonyl containing from one to six carbon atoms; R² is isobutylor 2-hydroxyisobutyl; X is —S(O)_(n)— or oxygen; R³ is selected from thegroup consisting of halogen, hydroxy, carboxyl, alkoxy, alkoxycarbonyl,—NR⁴R⁵ and —NR⁶(CH₂)_(m)NR⁴R⁵; each R⁴ and R⁵, which may be the same ordifferent, is independently: hydrogen; straight- or branched-chain alkylcomprising from one to six carbon atoms, optionally substituted by oneor more groups R⁷ which may be the same or different; straight- orbranched-chain alkenyl or alkynyl comprising from two to four carbonatoms; cycloalkyl containing from three to six carbon atoms optionallysubstituted by straight- or branched-chain alkyl containing from one tosix carbon atoms; phenyl optionally substituted by from one to fivegroups which may be the same or different selected from the groupconsisting of halogen, alkoxy, alkoxycarbonyl, amino, monoalkylamino anddialkylamino; a heterocyclic ring which may be saturated or unsaturatedcontaining five or six ring atoms and from one to three heteroatomswhich may the same or different selected from nitrogen, sulfur andoxygen; or R⁴ and R⁵, together with the nitrogen atom to which they areattached, form a saturated or unsaturated heterocyclic ring containingfrom four to six ring atoms, which ring may optionally contain anotherheteroatom selected from the group consisting of nitrogen, oxygen andsulfur and may be optionally substituted by from one to four groupswhich may be the same or different selected from the group consisting ofalkyl, phenyl and benzyl; R⁶ is hydrogen or straight- or branched-chainalkyl containing from one to six carbon atoms; R⁷ is selected from thegroup consisting of halogen, hydroxy, carboxyl, alkoxycarbonyl and—NR⁸R⁹; R⁸ and R⁹, which may be the same or different, each representhydrogen or straight- or branched-chain alkyl containing from one to sixcarbon atoms; n is zero, one or two; m is an integer from two to four;or a pharmaceutically acceptable salt or solvate thereof, in combinationwith a therapeutically effective amount of a second agent, or apharmaceutically acceptable salt or solvate thereof.
 2. The methodaccording to claim 1 in which the second agent is selected from thegroup consisting of modulators of NS3-4A protease, nucleoside modulatorsof NS5B RNA-dependent RNA polymerase, nonnucleoside modulators of NS5BRNA-dependent RNA polymerase and immunomodulatory agents.
 3. The methodaccording to claim 1, in which the second agent is a ketoamide inhibitorof NS3/4A protease.
 4. The method according to claim 3 in which thesecond agent is BILN 2061 or VX-950.
 5. The method according to claim 3in which the second agent is (1R, 2S,5S)-3-azabicyclo[3,1,0]hexane-2-carboxamide,N-[3-amino-1-cyclobutylmethyl)-2,3-dioxopropyl]-3-[(2S)-2-[[[1,1-dimethylethyl]amino]carbonylamino]-3,3-dimethyl-1-oxobutyl]-6,6-dimethyl.6. The method according to claim 1 in which the second agent isvalopicitabine or 7-Deaza-2′-C-methyl-adenosine.
 7. The method accordingto claim 1 in which the second agent is JTK-003, JTK-009, R803, HCV-371,HCV-086, HCV-796 or a benzo-1,2,4-thiadiazine.
 8. The method accordingto claim 1 in which the second agent is a small inhibitory RNA or ashort hairpin RNA capable of inhibiting HCV replication.
 9. The methodaccording to claim 1 in which the second agent is IFN, pegylated IFN orribavirin.
 10. The method according to claim 1 in which the second agentis IFNα, IFNα2a, IFNα2b or pegylated IFNα2a.
 11. The method according toclaim 10 further comprising the administration of ribavirin.
 12. Themethod according to claim 1 in which the second agent is PEGASYS® orPEG-INTRON®.
 13. The method according to claim 12 further comprising theadministration of ribavirin.
 14. The method according to claim 1 inwhich the second agent is a modulator of a Toll-like receptors (TLR).15. The method according to claim 1 in which the second agent is amodulator of TLR3, TLR7, TLR8 or TLR9.
 16. The method according to claim1 in which the second agent is a polynucleotide comprising unmethylatedCpG.
 17. The method according to claim 1 in which the second agent isANA975.
 18. The method according to claim 1 in which the second agent iscelgosivir.
 19. The method of claim 1 in which the second agent is areversible inhibitor of inosine monophosphate dehydrogenase.
 20. Themethod of claim 1 in which the second agent is ursodeoxycholic acid, ora derivative thereof.
 21. The method according to claim 1 in which saidadministration is sequential.
 22. The method according to claim 1 inwhich said administration is simultaneous.
 23. The method according toclaim 1 in which the cyclosporin derivative of formula (I) is3-[(R)-2-(N,N-dimethylamino)ethylthio-Sar]-4-(gamma-hydroxymethylleucine)cyclosporin,or a pharmaceutically acceptable salt or solvate thereof.
 24. The methodaccording to claim 1 in which the cyclosporin derivative is administeredorally.
 25. The method according to claim 1, in which the amount ofcyclosporin derivative administered is from about 1 to about 1000 mg perday.
 26. The method according to claim 22, in which the amount ofcyclosporin derivative administered is from about 25 to 150 mg per day.27. A composition comprising a cyclosporin derivative of general formula(I) as defined in claim 1 or a pharmaceutically acceptable salt orsolvate thereof, in combination with a therapeutically effective amountof a second agent, or a pharmaceutically acceptable salt or solvatethereof.
 28. A composition comprising3-[(R)-2-(N,N-dimethylamino)ethylthio-Sar]-4-(gamma-hydroxymethylleucine)cyclosporin,or a pharmaceutically acceptable salt or solvate thereof, in combinationwith a therapeutically effective amount of a second agent, or apharmaceutically acceptable salt or solvate thereof.